Controlling protein levels in eucaryotic organisms

ABSTRACT

The invention relates to novel compounds comprising a ubiquitination recognition element and a protein binding element. The invention also relates to the use of said compounds for modulating the level and/or activity of a target protein. The compounds are useful for the treatment of disease such as infections, inflammatory conditions, cancer and genetic diseases. The compounds are also useful as insecticides and herbicides.

FIELD OF INVENTION

[0001] The subject invention relates to novel compounds and their use incontrolling levels of proteins in eukaryotic organisms.

BACKGROUND OF INVENTION

[0002] Ubiquitin Mediated Protein Degradation

[0003] Ubiquitin is known to be one of several factors required forATP-dependent protein degradation in eukaryotic cells. One function ofintracellular protein degradation, most of which is ATP-dependent, isselective elimination of damaged and otherwise abnormal proteins.Another is to confer short half-lives on undamaged proteins whoseconcentrations in the cell must vary as functions of time, as is thecase, for example, with many regulatory proteins. Many other proteins,while long-lived as components of larger macromolecular complexes suchas ribosomes and oligomeric proteins, are metabolically unstable in afree, unassociated state. Ubiquitination is also involved in the controlof cell surface receptors such as platelet-derived growth factor (PDGF),the T cell receptor, G protein-coupled receptors and others. In additionto these proteins complexed with ubiquitin, ubiquitin is also foundcovalently linked to lipids in membranes (Guarino, L A, 1995, Cell 80,301-309).

[0004] Ubiquitin, a 76-residue protein, is present in eukaryotes eitherfree or covalently joined, through its carboxyl-terminal glycineresidue, to various cytoplasmic, nuclear, and integral membraneproteins. A family of ubiquitin-conjugating enzymes (also called E2enzymes) catalyzes the coupling of ubiquitin to such proteins(ubiquitination) generally in combination with a recognition elementcalled E3 that may also function to carry out the ubiquitination. Thefact that the protein of ubiquitin is conserved among eukaryotes to anextent unparalleled among known proteins suggests that ubiquitinmediates a basic cellular function.

[0005] It has been shown that selective degradation of many short-livedproteins requires a preliminary step of ubiquitin conjugation to atargeted proteolytic substrate. One role of ubiquitin is to serve as asignal for attack by proteases specific for ubiquitin-protein conjugates(Finley and Varshavsky, Trends Biochem. Sci. 10:343-348 (1985)).

[0006] At least some short-lived proteins are recognized as such becausethey contain sequences (degradation signals) which make these proteinssubstrates of specific proteolytic pathways. The first degradationsignal to be understood in some detail comprises two distinctdeterminants: the protein's amino-terminal residue and a specificinternal lysine residue, the N-end rule (Bachmair et al., Science234:179-186 (1986); Bachmair and Varshavsky, Cell 56:1013-1032 (1989)).The N-end rule, a code that relates the protein's metabolic stability tothe identity of its amino-terminal residue (Bachmair et al., Science234:179-186 (1986), is universal in that different versions of the N-endrule operate in all of the eukaryotic organisms examined, from yeast tomammals (Gonda et al., J. Biol. Chem. 264:16700-16712 (1989)).

[0007] The second essential determinant of the N-end rule-baseddegradation signal, referred to as the second determinant, is a specificinternal lysine residue in the substrate protein that serves as the siteof attachment of a multiubiquitin chain. Formation of the multiubiquitinchain on a targeted short-lived protein is essential for the protein'ssubsequent degradation. The enzymatic conjugation of ubiquitin to otherproteins involves formation of an isopeptide bond between thecarboxy-terminal glycine residue of ubiquitin and the epsilon-aminogroup of a lysine residue in an acceptor protein. In a multiubiquitinchain, ubiquitin itself serves as an acceptor, with several ubiquitinmoieties attached sequentially to an initial acceptor protein to form achain of branched ubiquitin-ubiquitin conjugates (Chau et al., Science243:1576-1583 (1989)).

[0008] The elucidation of the fundamental rules governing the metabolicstability of proteins in cells, and especially the deciphering of theN-end rule-based degradation signal, has made possible the manipulationof proteins to vary their half-lives in vivo (Bachmair and Varshavsky,Cell 56:1019-1032 (1989)).

[0009] The N-degron is an intracellular degradation signal whoseessential determinant is a specific (“destabilizing”) N-terminal aminoacid residue of a substrate protein. A set of N-degrons containingdifferent destabilizing residues is manifested as the N-end rule, whichrelates the in vivo half-life of a protein to the identity of itsN-terminal residue. The fundamental principles of the N-end rule, andthe proteolytic pathway that implements it, are well established in theliterature (see, e.g., Bachmair et al., Science 234: 179 (1986);Varshavsky. Cell 69: 725 (1992), U.S. Pat. Nos.: 5,122,463; 5,132,213;5,093,242 and 5,196,321) the disclosures of which are incorporatedherein by reference in their entirety.

[0010] In eukaryotes, the N-degron comprises at least two determinants:a destabilizing N-terminal residue and a specific internal lysineresidue (or residues). The latter is the site of attachment of amultiubiquitin chain, whose formation is required for the degradation ofat least some N-end rule substrates. Ubiquitin is a protein whosecovalent conjugation to other proteins plays a role in a number ofcellular processes, primarily through routes that involve proteindegradation.

[0011] In a stochastic view of the N-degron, each internal lysine of aprotein bearing a destabilizing N-terminal residue can be assigned aprobability of being utilized as a multiubiquitination site, dependingon time-averaged spatial location, orientation and mobility of thelysine. For some, and often for all of the Lys residues in a potentialN-end rule substrate, this probability is infinitesimal because of thelysine's lack of mobility and/or its distance from a destabilizingN-terminal residue.

[0012] It is possible to construct a thermolabile protein bearing adestabilizing N-terminal residue in such a way that the protein becomesa substrate of the N-end rule pathway only at a temperature high enoughto result in at least partial unfolding of the protein. This unfoldingactivates a previously cryptic N-degron in the protein by increasingexposure of its (destabilizing) N-terminal residue, by increasingmobilities of its internal Lys residues, or because of both effects atonce. Since proteolysis by the N-end rule pathway is highly processive,any protein of interest can be made short-lived at a high(nonpermissive) but not at a low (permissive) temperature by expressingit as a fusion to the thus engineered thermolabile protein, with thelatter serving as a portable, heat-inducible N-degron module.

[0013] The heat-inducible N-degron module can be any protein or peptidebearing a destabilizing N-terminal residue that becomes a substrate ofthe N-end rule pathway only at a temperature high enough to be useful asa nonpermissive temperature.

[0014] The idea of metabolically destabilizing a protein or peptide ofinterest using a protein or peptide (ie targeting a protein or peptidefor degradation) has been described in U.S. Pat. No. 5,122,463. Thismetabolic destabilization requires that the protein or peptide ofinterest must contain a second determinant of the N-end rule-baseddegradation signal. The method comprises contacting the protein orpeptide of interest with a targeting protein or peptide that interactsspecifically with the protein or peptide of interest. The targetingpeptide or protein is characterized as having a destabilizingamino-terminal amino acid according to the N-end rule of proteindegradation.

[0015] The ability to activate the ubiquitination and degradation ofother proteins not containing an N-terminus N-degron signal has beenshown in a multisubunit protein where the N-degron signals are locatedon different subunits and still target a protein for destruction (U.S.Pat. No. 5,122,463). Moreover, in this case (trans recognition) only thesubunit that bears the second N-degron signal (lysine) determinant isactually degraded. Thus, an oligomeric protein can contain bothshort-lived and long-lived subunits. In these examples thedemonstrations are all based on known multisubunit proteins andalterations of these to bring about the destabilization of subunitsinvolved in these multisubunit complexes.

[0016] A different aspect of targeting the ubiquitination system basedon chimeric proteins of E2 to achieve selective targeting andalterations in the levels of proteins has been described (Gosink M M andVierstra R D, 1995, Proc. Natl. Acad. Sci. 92, 9117-9121). Theseresearchers demonstrated that selective ubiquitination and degradationcan be achieved using a protein, which is a fusion protein of aubiquitinating protein with a binding protein.

[0017] In one interesting study of the N end rule, the degradation ofDHFR was stabilized by the binding of a small molecule indicating thatbinding small molecules could prevent the degradation of proteins. Thiswas also suggested in U.S. Pat. No. 5,122,463 where the idea of usingpeptides and proteins to target the ubiquitination of proteins to whichthey bind is suggested. In this patent the peptides are described asbinding in such a way that the peptide interferes with the folding ofthe target protein “folding-interfering targeting peptides” suggestingalso that peptides binding might prevent degradation as seen with DHFR.Indeed in this patent the focus for the peptides is the sequence of thetarget protein to give rise to these destabilizing residues.

[0018] Other Protein Covalent Modification for Protein Targeting

[0019] A number of systems mirror the protein modification pathway ofubiquitin. Among these are based on the attachment of Apg 12, Rub1/Nedd8and Smt3/SUMO-1 to proteins in addition to the ubiquitin pathway. Inthese systems homology at the level of sequence is seen but also clearparallels can be drawn based on the functional elements involved in thevarious systems (S Jentsch and H. D. Ulrich, Nature (1998) 395,321-322).

[0020] In the case of the Apg12 system this protein is involved in theautophagy of various cellular components. Apg12 appears to be thefunctional homologue of ubiquitin and is transferred via Apg7 and Apg 10the functional homologue of the E1 and E2 ubiquitin conjugating enzymes,respectively. Apg12 transferred via Apg7 and Apg10 is used to modifyApg5 to activate autophagy. The analysis of the sequence of Apg7 shows aconsiderable homology to the E1 enzymes of the ubiquitin pathway. In thecase of Rub1/Nedd8 system this protein is involved in some regulatoryrole. The Smt3/SUMO-1 system is involved in the targeting of proteins.

[0021] Drug Targets

[0022] The number of drug targets for human therapeutics is around 400human gene products, such as enzymes, receptors and ion channels. Butthere may be 2500-5000 molecular targets whose exploitation may becapable of restoring function in the 100 or so common human polygenicdiseases. Many of these new targets are being discovered by theintensive search of the human genome by various groups using focused andrandom methods.

[0023] The following are examples of drug targets which are the subjectof investigation by various pharmaceutical companies: B7.1 and B7,TNFR1m(p55), TNFR2 (p75), NADPH oxidase, Bcl/Bax and other partners inthe apotosis pathway, C5a receptor, HMG-CoA reductase, PDE Vphosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDEII,PDEIII, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide (NO)synthase, cyclo-oxygenase 1, cyclo-oxygenase 2, 5HT receptors, dopaminereceptors, G proteins ie Gq, histamine receptors, 5-lipoxygenase,tryptase serine protease, thymidylate synthase, purine nucleosidephosphorylase, GAPDH trypanosomal, glycogen phosphorylase, Carbonicanhydrase, chemokine receptors, JAK/STAT, RXR and similar, HIV 1protease, HIV 1 integrase, influenza, neuraminidase, hepatitis B reversetranscriptase, sodium channel, multi drug resistance (MDR), proteinP-glycoprotein (and MRP), tyrosine kinases, CD23, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR, ICAM1, Ca++channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L, neurokinins andreceptors, inosine monophosphate dehydrogenase, p38 MAP Kinase,Ras/Raf/MEK/ERK pathway, interleukin-1 converting enzyme, caspase, HCV,NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-1),protease, cytomegalovirus (CMV) protease, poly (ADP-ribose) polymerase,cyclin dependent kinases, vascular endothelial growth factor, oxytocinreceptor, microsomal transfer protein inhibitor, bile acid transportinhibitor, 5 alpha reductase inhibitors, angiotensin II, glycinereceptor, noradrenaline reuptake receptor, endothelin receptors,neuropeptide Y and receptor, adenosine receptors, adenosine kinase andAIMP deaminase, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7),farnesyltransferases, geranylgeranyl transferase, TrkA a receptor forNGF, beta-amyloid, tyrosine kinase Flk-1/KDR, vitronectin receptor,integrin receptor, Her-2/neu, telomerase inhibition, cytosolicphospholipase A2, EGF receptor tyrosine kinase.

[0024] Insecticide target examples include, ecdysone 20-monooxygenase,ion channel of the GABA gated chloride channel, acetylcholinesterase,voltage-sensitive sodium channel protein, calcium release channel, andchloride channels.

[0025] Herbicide target examples include Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

[0026] These various targets are typically used in screens that look fora compound to alter the level of activity of the selected target andrequire the compound to be in solution. In some cases the assay todetermine activity in a potential compound has to be based on a cellbased assay. The best assays for compound screens are where theinteraction of two molecules is modulated allowing the development ofrapid assays based on the determination of binding.

[0027] In addition to the drawbacks of current drug and compounddiscovery efforts described above, problems of specificity arise due tothe common basis for the activity of various compounds. For example intrying to find compounds which block the dopamine receptor, one isinterested in the inhibition of a specific receptor sub-type due to itsexpression in a selected tissue. The binding site of the receptor isdesigned to bind to dopamine and thus has a common structure across thevarious sub-types of receptors. This homology of structure at the targetsite of the discovery effort makes it difficult to identify compoundswith optimal levels of specificity for given sub-types and thusdifficult to achieve the levels of therapeutic affect desired.

[0028] The present invention provides a solution to this problem.

[0029] Antigen Presentation

[0030] The target degradation of various proteins in the cell is amechanism for the presentation of various peptides in the context ofMHC. It has been demonstrated that the ubiquitination of intracellularproteins leads to the degradation of the protein via the 26S proteasomeand enhanced presentation of the resultant peptides in the context ofMHC I. This enhanced presentation leads to improved immune responses bythe stimulation of various cells involved in the immune system. In manydiseases the antigenicity of various proteins does not appear to bepotent enough to generate a robust immune response. For example in thecase of cancer certain antigens are present but fail to elicite a potentimmune response (Tobery T and Siliciano R F., 1999, J Immunol. 162,639-642). The present invention provides a solution to this problem ofgenerating an improved immune response.

[0031] Antisense

[0032] Antisense technology is a novel drug therapy approach. Antisensedrugs work at the genetic level to interrupt the process by whichdisease causing proteins are produced. Proteins play a central role invirtually every aspect of human metabolism. Many human diseases are theresult of inappropriate protein production. Antisense drugs are designedto inhibit the production of disease causing proteins. These antisensedrugs function by binding to specific nucleic acid sequences in a celland block the production of specific proteins in this way a specificproteins level is reduced. Examples of targets for this technology arevirus-based diseases, cancer, Crohn's disease, renal transplantrejection, psoriasis, ulcerative colitis, and inflammation. The specifictargets are; HPV, HIV, CMV, hepatitis C, ICAM- 1, PKC-alpha, c-rafkinase, Ha-ras, TNF-alpha and VLA-4.

SUMMARY OF INVENTION

[0033] The invention comprises compositions and methods for controllingthe levels of proteins in eukaryotic organisms. This control of proteinlevels is achieved using an exogenous molecule able to affectubiquitination of a given protein. The ubiquitinated protein is targetedfor intracellular degradation via normal cellular pathways. Theexogenous molecule able to selectively target ubiquitination of apre-selected protein comprise; a ubiquitination recognition element andtarget protein binding element for a pre-selected protein covalentlylinked to form the compositions of the invention.

[0034] The ubiquitination recognition element is designed to interactwith the ubiquitination mechanisms of the cell allowing theirrecruitment. The target protein binding element binds to pre-selectedprotein in order to effectively present the ubiquitin recognitionelement.

[0035] This invention offers a number of improvements over the artespecially for drug development. The invention provides a more costeffective route for drug development and drugs with improved activity.

[0036] The invention comprises compounds for activating theubiquitination of a target protein comprising, a ubiquitinationrecognition element which is able to bind to either the E3 or E2functional elements of the ubiquitination system, the ubiquitinationrecognition element has a molecular weight less than 30,000 and has abinding affinity for the E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹ and; a target protein binding element that isable to bind specifically to a target protein, the target proteinbinding element has a molecular weight of less than 30,000 and has abinding affinity for the target protein greater than 10⁵ M⁻¹, theubiquitination recognition element is covalently linked to the targetprotein binding element.

[0037] The invention also comprises compounds for activating theubiquitination of a target protein comprising, a ubiquitinationrecognition peptide element which is able to bind to either the E3 or E2functional elements of the ubiquitination system, the ubiquitinationrecognition peptide element has a molecular weight less than 30,000 andhas a binding affinity for the E3 and/or E2 elements of theubiquitination system of at least 10² M⁻¹ and a target protein bindingelement that is able to bind specifically to a target protein, thetarget protein binding element has a molecular weight of less than30,000 and has a binding affinity for the target protein greater than10⁵ M⁻¹, the ubiquitination recognition peptide element is covalentlylinked to the target protein binding element.

[0038] The invention also comprises compounds for activating theubiquitination of a target protein comprising, a ubiquitinationrecognition element which is able to bind to either the E3 or E2functional elements of the ubiquitination system, the ubiquitinationrecognition element has a molecular weight less than 30,000 and has abinding affinity for the E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹ and a target protein binding peptide elementthat is able to bind specifically to a target protein wherein the targetprotein peptide binding element has a molecular weight of less than30,000 and has a binding affinity for the target protein greater than10⁵ M⁻¹, wherein the ubiquitination recognition element is covalentlylinked to the target protein binding peptide element.

[0039] The invention comprises compounds for activating theubiquitination of a target protein comprising, a ubiquitinationrecognition peptide element which is able to bind to either the E3 or E2functional elements of the ubiquitination system, wherein theubiquitination recognition peptide element has a molecular weight lessthan 30,000 and has a binding affinity for the E3 and/or E2 elements ofthe ubiquitination system of at least 10² M⁻¹ and a target proteinbinding peptide element that is able to bind specifically to a targetprotein wherein the target protein binding peptide element has amolecular weight of less than 30,000 and has a binding affinity for thetarget protein greater than 10⁵ M⁻¹ where the ubiquitination recognitionpeptide element is covalently linked to the target protein bindingpeptide element.

[0040] The invention also provides a method of modulating the leveland/or activity of at least one target protein in an eukaryotic cell viathe modulation of ubiquitination of the at least one target proteincomprising contacting the cell with a compound comprising; aubiquitination recognition element which is able to bind to either theE3 or E2 elements of the ubiquitination system, wherein theubiquitination recognition element has a molecular weight less than30,000 and has a binding affinity for the E3 and/or E2 elements of theubiquitination system of at least 10² M⁻¹ and; a target protein bindingelement that is able to bind specifically to a target protein whereinthe target protein binding element has a molecular weight of less than30,000 and has a binding affinity for the target protein greater than10⁵ M⁻¹, the ubiquitination recognition element is covalently linked tothe target protein binding element.

[0041] The invention also provides a method of treating an infection ina mammal comprising administering to the mammal an amount of a compoundsufficient to eliminate and/or reduce the infection comprisingcontacting the mammal with a compound comprising; a ubiquitinationrecognition element which is able to bind to either the E3 or E2elements of the ubiquitination system, wherein the ubiquitinationrecognition element has a molecular weight less than 30,000 and has abinding affinity for the E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹ and; a target protein binding element that isable to bind specifically to a target protein wherein the target proteinbinding element has a molecular weight of less than 30,000 and has abinding affinity for the target protein greater than 10⁵ M⁻¹, whereinthe ubiquitination recognition element is covalently linked to thetarget protein binding element.

[0042] The invention is also a method of treating cancer or tumor in amammal comprising administering to the mammal an amount of a compoundsufficient to reduce the size of the tumor comprising contacting themammal with a compound comprising; a ubiquitination recognition elementwhich is able to bind to either the E3 or E2 elements of theubiquitination system, wherein the ubiquitination recognition elementhas a molecular weight less than 30,000 and has a binding affinity forthe E3 and/or E2 elements of the ubiquitination system of at least 10²M⁻¹ and; a target protein binding element that is able to bindspecifically to a target protein wherein the target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for the target protein greater than 10⁵ M⁻¹, wherein theubiquitination recognition element is covalently linked to the targetprotein binding element.

[0043] The invention also provides a method of generating a compoundwhich comprises covalently linking a target protein binding element to aubiquitination recognition element.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 shows the basic elements of the invention, where a moleculecontaining a ubiquitination recognition element and a target proteinrecognition element brings together the target protein and aubiquitination system to stimulate the ubiquitination of the targetprotein by the ubiquitination system.

[0045]FIG. 2 shows the synthetic steps for synthesis of L-chicoric acid.

[0046]FIG. 3 shows the synthetic steps for synthesis of N-bromoacetylethylenediamine

[0047]FIG. 4 shows the synthetic steps for synthesis of bromoacetylatedL-chicoric acid.

[0048]FIG. 5 shows the conjugation of the ubiquitination recognitionelement to L-chicoric acid.

[0049]FIG. 6 shows the synthetic steps for synthesis of ubiquitinationrecognition element linked to glutathione.

[0050]FIG. 7 shows the synthetic steps for synthesis of ubiquitinationrecognition element linked to fluorescein.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention relates to a method of development ofcompounds which are active via a new mechanism of action. The inventionis to a new class of molecules that make use of the targetedmodification and/or degradation of proteins to modulate a selectedtarget protein's concentration and/or activity. This is achieved throughthe construction of a bi-functional molecule. This control of proteinlevels is achieved using an exogenous molecule able to affectubiquitination of a given protein. The ubiquitinated protein is targetedfor intracellular degradation via normal cellular pathways.

[0052] The exogenous molecules able to selectively target ubiquitinationof a pre-selected protein comprise; a ubiquitination recognition elementand target protein binding element for a pre-selected protein covalentlylinked to form the compositions of the invention (FIG. 1).

[0053] The ubiquitination recognition element is designed to interactwith the ubiquitination mechanisms of the cell allowing theirrecruitment. The target protein binding element binds to a pre-selectedprotein in order to effectively present the ubiquitin recognitionelement.

[0054] Definitions

[0055] Ubiquitin, as used herein is a protein which is functionally andstructurally related to cellular ubiquitin. The functionally activity isdefined via its conjugation to other proteins forming covalent proteinconjugates through the action of an ATP dependent cellular pathway andits protein sequence. The structural relation is defined either bysequence homology and/or structure homology. Sequence homology isdefined by a BLAST sequence homology analysis (Altschul S F et al., JMol Biol 1990, 215, 403-410) where the E value is less than 0.063,representing a significant homology. Structural homology is defined by aVAST homology analysis where the p-value is less than 0.0001,representing a significant homology.

[0056] Ubiquitination, as used herein is the formation of a covalentbond between a cellular protein and a ubiquitin protein (as definedabove) through the action of an ATP dependent cellular pathway.

[0057] Ubiquitination system, as used herein is a cellular system ableto direct the formation of covalent protein conjugates between ubiquitinand other proteins. Ubiquitination systems consist of one or a number ofproteins involved in the activation of ubiquitin, recognition of aprotein for ubiquitination and formation of ubiquitin:proteinconjugates.

[0058] Ubiquitination recognition element, as used herein is a chemicalmoiety which is able to bind with a ubiquitination systems proteins orits component proteins. This binding is further defined by the abilityof the chemical moiety to promote the ubiquitination of a proteinattached directly or indirectly to the moiety.

[0059] Ubiquitination recognition peptide element, as used herein is apeptide moiety which is able to bind with a ubiquitination systemsproteins (other than those of the N-end rule) or its component proteins.This binding is further defined by the ability of the peptide moiety topromote the ubiquitination of a protein attached directly or indirectlyto the moiety.

[0060] Ubiquitination recognition site, as used herein is a sequence ofa protein which is known to act as the recognition site forubiquitination systems. This ubiquitination recognition site is furtherdefined by the ability of the site to promote the ubiquitination of aprotein attached directly or indirectly to the site.

[0061] Target protein, as used herein is a protein selected forubiquitination using a compound of the subject invention.

[0062] Target protein binding element, as used herein is a chemicalmoiety which is able to bind to a target protein. Examples of thesebinding elements include drugs and toxin molecules.

[0063] Target protein binding peptide element, as used herein is apeptide structure which is selected to bind to a target protein.

[0064] The means by which the compositions of the invention areidentified and synthesized is described below.

[0065] The invention solves problems of library construction andscreening by making use of the binding activity of a small molecule todevelop biologically active and valuable molecules. This removes theproblems associated with the synthesis of chemical libraries in that a)the compounds can be screened bound to solid phase (in fact an advantageof the subject invention) and b) the presence of a linker element is autility of the subject invention which is commonly a problem in solidphase chemistries. These specific advantages in combination allow for anoptimal route to the generation of chemical libraries and theirscreening. These two elements combine synergistically resulting in rapiddrug development. In addition to these advantages, the hit rates foractive compounds is increased as generalized binding is optimal, notjust binding to the active site which limits the potential drugcompounds which may be found following conventional drug screeningapproaches. The invention also allows for the development of smallmolecule drugs whose development is problematic using traditionalmethods, for example finding a small molecule which can block theinteraction of two large proteins such as is seen with cell cellinteractions, some receptor ligand interactions, and intra cellularsignaling pathways.

[0066] Since the method of the subject invention does not make use ofthe ‘active’ site of a given target protein, it is able to achieve alevel of specificity for a drug molecule previously considered extremelydifficult and uncertain using conventional drug discovery efforts. Thisadvantage stems from the constraints placed on existing drug discoveryefforts that are based on the need to inhibit an enzyme or receptorbinding site that is common to a series of different proteins indifferent tissues and with very different roles in the physiology of theorganism. These constrains are based on the common structural elementsin the binding or catalytic sites of these related proteins which formthe site for conventional drug discovery. The common structural elementstypically result in the selection of drugs that will inhibit the wholeseries of different proteins as these structural elements form the basisfor the binding of the drug molecules selected from the screen. Thusconventional drug screening approaches result in the selection of drughits which do not provide the degree of selectivity desired to bringabout a desired therapeutic affect. In the subject invention, since theactive site does not need to be the target for the selection ofmolecules that form the basis of the drug molecule, a significantimprovement in the discovery of highly selective drugs is achieved. Theconsequence is the development of drugs with an enhanced therapeuticvalue. This advantage is further enhanced by the ability of this drugdiscovery approach to make use of the whole surface of the given proteintarget to find molecules with the desired binding specificity. Thisadvantage is then combined with the ability to make use of a rapidscreen that is wholly based on the use of binding and thus achieves alevel of speed and through put not possible with other methods. Thisadvantage is of great value when the desire is to find a very specificinhibitor of a given member of a protein family that is highlyhomologous and thus extremely difficult or impossible for drug discoverybased on the effector, receptor or catalytic site of the given protein.This invention thus provides a means for the development of compounds ofthe invention which are variously; therapeutics, have variouspharmacological activities, herbicides, pesticides, insecticides,antivirals, antifungals, anti-parasitics and are able to selectivelymodify the performance of an organism.

[0067] The subject invention also includes a method to enhance theimmunogenicity of a given protein. This is achieved by enhancing thedegradation of a given protein via the 26S proteasome by selectiveubiquitination resulting in increased presentation of the antigen aspeptides in the context of MHC I. The ability to enhance theimmunogenicity of a given protein has great value in the treatment ofinfectious diseases (HIV, HBV, HCV, Herpes, etc) and also in thetreatment of cancer where the cancer antigen is not very immunogenic.The use of this approach for cancer treatment in combination with cancervaccine approaches and/or use of cytokines such as gamma interferon, isalso contemplated.

[0068] The subject invention also provides a method whereby a smallmolecule is used to regulate the levels of a protein geneticallyengineered into a cell line or organism. This is achieved via themodification of a gene encoding the protein of interest to contain, inaddition to the desired activity of the protein, a binding site for asmall molecule able to activate targeted covalent modification. Thismodified nucleic acid encoding an protein is then used to generate agenetically engineered cell or organism. This approach allows for thespecific modulation of a given proteins action after the production of agenetically modified cell or organism on addition of compounds of theinvention able to activate targeted covalent modification.

[0069] The subject invention also permits the development of specificcompounds of the invention which can be used to target specific proteinsfor degradation to allow the determination of a given proteins rolewithin the cell or organism. This approach is useful in targetvalidation for the development of pharmaceuticals, for conducting basicresearch and for target validation for may other discovery effortsdirected to the discovery of molecules able to bring about modulation ofan amino acids levels and/or function.

[0070] Target Protein Binding Elements

[0071] The target protein binding elements of the invention aremolecular structures which bind target proteins, and are used in thecompounds of the invention to target the ubiquitination recognitionelements to the target protein. These target protein binding elementsare covalently linked to the ubiquitination recognition elements to formthe compounds of the invention and provide the linkage between these twoelements of the compounds of the invention. When the target proteinbinding element of a compound of the invention binds to a given targetprotein it presents the ubiquitination recognition element to allow theactivation of the ubiquitination pathway and subsequent ubiquitinationof the target protein bound by the target protein binding element.

[0072] Target protein binding elements are small organic moleculesdefined by binding to a predetermined target molecule, having amolecular weight from 50 to 30,000 and with a binding affinity ofgreater than 10⁵ M⁻¹ for the target protein of interest. The bindingaffinity in an advantageous embodiment is greater than 10⁶ M⁻. Themolecular weight in an advantageous embodiment is between 50 and 3,000.The binding affinity in a more advantageous embodiment is greater than10⁸ M⁻¹. The molecular weight in a more advantageous embodiment isbetween 100 and 2,000. Most drugs are typically either neutral, weakacids or bases. Examples of known specific drugs are phenytoin (pKa of8.3) and aspirin (pKa of 3.0).

[0073] Also target protein binding elements can be selected based onhaving at least one the following characteristics; less than 50 H-bonddonors, MW less than 5,000, ClogP or MLogP (calculated log P, based onthe Pomona College Medicinal Chemistry program ClogP or using MolecularDesign Limited MACCS and ISIS based programs MlogP, logP (the logarithmof the octanol/water partition coefficient) less than 6, sum of N's andO's (a rough measure of H-bond acceptors) less 100.

[0074] Also target protein binding elements can be selected based havingon at least one the following characteristics; less than 5 H-bonddonors, MW less than 500, ClogP or MLogP less than 5, sum of N's and O's(a rough measure of H-bond acceptors) less 10.

[0075] Also target protein binding elements can be selected based onhaving two or more combinations of the following characteristics; lessthan 5 H-bond donors, MW less than 500, ClogP or MLogP less than 5, sumof N's and O's (a rough measure of H-bond acceptors) less 10 (Lipinski CA, 1997, Adv. Drug Delivery Rev. 23, 3-25).

[0076] These target protein binding elements are different frompeptides, proteins and DNA and RNA in that they are not highly chargedor polar, are readily absorbed into the body due to the size andhydrophobicity. Also one of the other key properties of target proteinbinding elements is the stability relative to proteins which are stablewithin narrow ranges of temperature, pH and ionic strength due to theneed to maintain a give structural conformation of the foldedpolypeptide chain. Peptides although not as sensitive to the physicalproperties of an environment are relatively unsuitable as drugs due tothe poor biological stability, short half-life and poor bioavailabilitywithin cells and are not considered compounds of the invention.

[0077] Some examples of molecules which have moieties desired in atarget protein binding element include drug molecules and moleculesselected for binding and/or inhibition of various proteins functions,for example; fluorescein, biotin, antigens, L-deprenyl, Omeprazole,Clavulanate, organoarsenical compounds such as4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein, p-aminophenylarsineoxide, p-aminophenylarsine oxide, chicoric acid, captopril, enalapril,lovastatin, proscar, indinivar, zileuton, L-372,460 (J. Med Chem 41,401, 1998), apomorphine, N-n-propylnorapomorphine, dihydrexidine,quinpirole, clozapine, haloperidol, nitrocaramiphen, and iodocaramiphen.

[0078] It is evident from the small sample above that numerous examplesexists of chemistries which could form the basis of chemistries fortarget protein binding element. Also the numerous nature of thesepotential target protein binding elements is illustrative of thepotential ease with which such moieties can be discovered using routineexperimentation.

[0079] Compounds of the invention include small molecules used inveterinary, agricultural, food and environmental applications where abiological effect is generated. Examples of compounds of the inventionare fungicides, herbicides, pesticides, algaecides, insecticides,anti-virals, anti-parasitics etc. In addition compounds of the inventionare also molecules able to form covalent bonds with the target proteinsof interest, such as suicide inhibitors. Examples of well know drugsable to from covalent bonds, are as follows; L-deprenyl (Gerlach, M etal 1992, Eur. J. Pharmacol. 226, 97-108), Omeprazole (Howden, C W. 1991,Clin. Phamacokinet, 20, 38-49) and Clavulanate (Neu, H C. 1990, J. Am.Acad. Dermatol, 22, 896-904). In addition to these well known moleculesare a considerable number of other small molecules known to formcovalent bonds specifically with various proteins. Also consideredcompounds of the invention are enzyme substrates that are used tocovalently modify proteins (such as farnesylation, phosphorylation,glycosylation, and gerenylation), where the natural enzyme substrate ismodified in such a way that it contains a ubiquitination recognitionelement.

[0080] Target Protein Binding Peptide Elements

[0081] The target protein binding peptide elements of the invention arepeptide structures which are selected to bind to target proteins and areused in the compounds of the invention to target the ubiquitinationrecognition elements, excluding those based on the N-end rule, to thetarget protein. These target protein binding peptide elements arecovalently linked to the ubiquitination recognition elements, excludingthose based on the N-end rule to form the compounds of the invention andprovide the linkage between these two elements of the compounds of theinvention. When the target protein binding peptide element of a compoundof the invention binds to a given target protein it presents theubiquitination recognition element to allow the activation of aubiquitination pathway (not based on the N-end rule) and subsequentubiquitination of the target protein bound by the target protein bindingpeptide element. Examples of these are peptide selected fromcombinatorial libraries such as those expressed on the surface of phage(Yanofsky S D et al., Proc. Natl. Acad. Sci USA 1996, 93, 7381).Examples of target protein binding peptide elements include;

[0082] epsilon-aminocaproic acid-phospho-Y-E-E-I (SEQ ID #56) binding tosrc SH2 domain;

[0083] DREGCRRGWVGQCKAWFN (SEQ ID #57) binding to erythropoietin;

[0084] ETPTFTWEESNAYYWQPYALPL (SEQ ID #58) binding to IL-1alpha;

[0085] TFVYWQPYALPL (SEQ ID #59) binding to IL-1alpha;

[0086] VSLARRPLPPLPGGK (SEQ ID #60) binding to the SH3 domains of Src,Fyn, Lyn, Yes, PI3K;

[0087] KGGGAAPPLPPRNRPRL (SEQ ID #61) binding to the SH3 domains of Src,Fyn, Lyn, Yes;

[0088] AECHPQGPPCIEGRK (SEQ ID #62) binding to streptavidin;

[0089] GACRRETAWACGA (SEQ ID #63) binding to alpha5beta1 integrin;

[0090] DITWDQLWDLMK (SEQ ID #64) binding to E-selectin;

[0091] RNMSWLELWEHMK (SEQ ID #65) binding to E-selectin;

[0092] Targets of the Target Protein Binding Element

[0093] Targets of the target protein-binding element are numerous andare selected from proteins and proteins that are expressed in a cellsuch that at least a portion of the sequences is available within thecell. The term protein includes all sequences of amino acids greaterthan two and includes peptides. Below is a partial list of targetproteins. Any protein in eukaryotic cells are targets for ubiquitinationmediated by the compounds of the invention. Those of special interestare those which are involved in diseases or disease processes included;are infectious diseases of viral, microbial, and parasitic nature,metabolic diseases, aging, environmental diseases, genetic diseases,life style diseases. Also protein targets which are involved inperformance enhancement are also targets, such as those involved ingrowth and development, memory, and sensory perception.

[0094] Examples of viruses contemplated as targets of the subjectinvention are HIV1, HIV2, HLTV, CMV, HPV, HSV, hepatitis, HBV, HCV, HAV,HDV, HGV, influenza A, influenza B, influenza C, rhinoviruses,rotaviruses, entroviruses, Ebola, polio, chicken pox, RSV, coronavirus,adenoviruses, parainfluenza 3, coxsackie A, and epstein-barr virus.

[0095] The following are example of targets of the target proteinbinding elements of the subject invention, which include:

[0096] Receptors

[0097] CD124, B7.1 and B7, TNFR1m(p55), TNFR2 (p75), Bcl/Bax and otherpartners in the apotosis pathway, C5a receptor, CXCR1, CXCR2, 5HTreceptors, dopamine receptors, G proteins, ie Gq, histamine receptors,chemokine receptors, JAK/STAT cf ligand, RXR and similar, CD4, CD5, IL-2receptor, IL-1 receptor, TNF-alphaR, ICAM1, VCAM, VLA-4 integrin,selectins, CD40/CD40L, neurokinins and receptors, Ras/Raf/MEK/ERKpathway, vascular endothelial growth factor, oxytocin receptor,microsomal transfer protein inhibitor, angiotensin II, glycine receptor,noradrenaline reuptake receptor, endothelin receptors, neuropeptide Yand receptor, adenosine receptors, purinergic receptors (P2Y1, P2Y2,P2Y4, P2Y6, P2X1-7), TrkA a receptor for NGF, beta-amyloid, tyrosinekinase Flk-1/KDR, vitronectin receptor, integrin receptor, Her-2/neu,MCH receptor, IL-4 receptor alpha chain and the Toll-like receptors andhuman homologue, FKHR and AFX or the human homologues of daf2, daf16 andage1.

[0098] Enzymes

[0099] NADPH oxidase, HMG-CoA reductase, PDE V phosphodiesterase type,PDE IV phosphodiesterase type 4, PDE I, PDEII, PDEIII, squalene cyclaseinhibitor, nitric oxide (NO) synthase, cyclo-oxygenase 1,cyclo-oxygenase 2, 5-lipoxygenase, tryptase serine protease, thymidylatesynthase, purine nucleoside phosphorylase, GAPDH trypanosomal, glycogenphosphorylase, Carbonic anhydrase, HIV 1 protease, HIV 1 integrase,influenza, neuraminidase, hepatitis B reverse transcriptase, tyrosinekinases, CD23, tyrosine kinase p56 1ck, inosine monophosphatedehydrogenase, p38 MAP Kinase, interleukin-1 converting enzyme, caspase,HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotideformyl transferase, rhinovirus 3C protease, herpes simplex virus-1(HSV-1) protease, cytomegalovirus (CMV) protease, poly (ADP-ribose)polymerase, cyclin dependent kinases, 5 alpha reductase inhibitors,adenosine kinase and AMP deaminase, farnesyltransferases, geranylgeranyltransferase, telomerase, cytosolic phospholipase A2, EGF receptortyrosine kinase.

[0100] Membrane Transporters

[0101] Sodium channel, Ca⁺⁺ channels, multi drug resistance (MDR),protein P-glycoprotein (and MRP), bile acid transporter.

[0102] Insecticide target examples include, ecdysone 20-monooxygenase,ion channel of the GABA gated chloride channel, acetylcholinesterase,voltage-sensitive sodium channel protein, calcium release channel, andchloride channels.

[0103] Herbicide target examples include Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

[0104] Targets for anti-parasitic drug development include: Leishmania,proteins of the sterol synthesis pathway: Plasmodium, dihydrofolatereductase; dihydrofolate reductase-thymidylate synthase (bifunctional)resistance known due to mutations in the gene for this enzyme, hemepolymerase: Trypanosoma, ornithine decarboxylase, trypanothionereductase, Ornithine decarboxylase of the trypanosoma represents anideal candidate for destruction due to its long half-life and low turnover in trypanosoma. It has also been suggested that the shikimatepathway, which is a target for herbicide development, would also be ofvalue in the development of anti-parasitics for parasites of the phylumApicomlexa (ie. Plasmodium falciparum, Cryptosporidium parvum andToxoplasma gondii) as it absent from mammals.

[0105] Also considered targets of the subject invention are proteinsthat are involved in the performance of a cell and/or organism.Performance characters of a cell and/or organism are those characterswhich are considered desirable traits which a cell and/or organism hasin some part. These performance characters may be present in other cellsor organisms and be desired in cells and/or organisms which do notposses them. Examples of what are considered performance characters are,flower color, fragrances, specific shapes and colors in organisms such acats and dogs, disease resistance, growth rates, size, taste, alcoholyield from yeast. Thus performance characters are generally things thatare desired in cells and organisms used either in the production ofdesired products or in the production of esthetical value (look, taste,feel, smell and sound).

[0106] In the case of flower color (considered an esthetical value), theproteins involved in the biosynthesis of flavonoids, carotenoids andanthocyanins; including flavanone 3-hydroxylase, anthocyanin synthase,dihydroflavonol 4-reductase, flavonoid 3′, 5′-hydroxylase, anthocyanin5-aromatic acyltransferase, UDP-glucose: flavonoid3-O-glucosyltransferase, anthocyanin rhamnosyltransferase, anthocyanin3′-methyltransferase, anthocyanin 3′5′-methyltransferase,leucoanthocyanidin dioxygenase, anthocyanidin synthase, anthocyaninacyltransferase, chalcone synthase, chalcone flavanone isomerase,glutathione S-transferase, one considered as targets of the subjectinvention involved in performance characteristics. In addition to theproteins involved in the synthesis of flower color, the proteinsinvolved in the regulation of the expression of the synthases and otherproteins involved in the production of flower color are also consideredtargets of the subject invention. Examples of the regulatory genesinclude the R and C1 gene families, an2 and jaf13, the delila gene.Quattrocchio F. 1998,Plant J. 13(4),475-488.

[0107] Other potential target molecules of the subject invention includetargets as described above but also targets in all eukaryotic organisms.Potential targets exist in agriculture, veterinary and environmentalfields. For example in the agricultural field, molecules which areselective in action and non-toxic are highly desirable for use asherbicides, anti-virals, anti-parasitics, growth modulators and drugs;thus target molecules can be selected from certain animals, plants,viruses and parasites of interest to agriculture. In the veterinaryfield anti-virals, anti-parasitics, antibiotics, growth modulators,anti-inflammatory and drugs are of interest and target molecules can beselected from certain animals, viruses and parasites of interest inveterinary science. In the environmental field the potential targets arethe same as for agricultural but the aims are to control selectivelycertain populations either positively as in the case of an endangeredspecies but also negatively where a population has expanded itsenvironment or where a foreign organism is undesirable to a givenecosystem. Thus it is understood by those skilled in the art that theability to modulate the level of a selected target molecule could havewide ranging effects in very diverse areas of science, technology andhuman endeavors.

[0108] Ubiquitin

[0109] In this invention ubiquitin includes ubiquitin and ubiquitin likesequences related either by sequence homology, by structural homology orfunctional homology or having been described as related to ubiquitin inthe scientific literature. Functional homology to ubiquitin is definedbased on a proteins ability to be attached covalently to other proteinsvia an ATP dependent enzyme system, proteins transferred in this way areconsidered to be ubiquitin in this invention and the protein couplingstep is considered to be ubiquitination. In the case of sequencehomology protein sequences with a BLAST (Altschul S F et al., J Mol Biol1990, 215, 403-410) E (Expected) value of 0.063 or less are consideredin this invention to be ubiquitin. The BLAST search being run the NIHweb server (NIH, http://www.ncbi.nlm.gov). The E value is a parameterthat describes the chances of finding a sequence match based on chance.Thus the smaller the value the smaller the chance that the matchoccurred by chance. In the case of structural homology these aredetermined based on the VAST (NIH, http://www.ncbi.nlm.gov) analysis toyield p-value of less than 0.0001 are considered to be ubiquitin in thisinvention. The VAST (NIH, http://www.ncbi.nlm.gov) p-value is a measureof the significance of a comparison, expressed as a probability. Forexample if the p-value is 0.0001 then the odds are 10,000 to 1 againstseeing a match by chance.

[0110] A number of systems are also considered to be ubiquitinationpathways in this invention as these protein modification pathways,involved in the attachment of Apg12, Rub1/Nedd8 and Smt3/SUMO-1 aregenerally considered as being equivalent to the ubiquitin pathway due totheir functional homology to the ubiquitination pathway.

[0111] In these systems homology at the level of sequence is seen butalso clear parallels can be drawn based on the functional elementsinvolved in the various systems (S Jentsch and H. D. Ulrich, Nature(1998) 395, 321-322).

[0112] In the case of the Apg12 system this protein is involved in theautophagy of various cellular components. Apg12 appears to be thefunctional homologue of ubiquitin and is transferred via Apg7 and Apg10the functional homologue of the E1 and E2 conjugating enzymes of theubiquitin and final is used to modify Apg5 to activate autophagypossible via a targeting mechanism. The analysis of the sequence of Apg7shows a considerable homology to the E1 enzymes of the ubiquitinpathway.

[0113] In the case of Rub1/Nedd8 system these proteins are involved in aregulatory role. The Smt3/SUMO-1 system is also involved in thetargeting of proteins.

[0114] Antigens

[0115] Antigens of this invention are considered to be target proteinsof the subject invention. Antigens of the invention are proteins thatare derived from numerous sources, examples of which are intracellularproteins of the host, or other target organisms. The antigens can alsobe derived from other organisms and are presented intracellularly withinthe organism of interest. Typically these antigens are derived from aninfectious organism such as a virus, bacteria or fungi or derived from anormal protein or one mutated in a given disease tissue. In oneembodiment of the invention antigens present in cancer cells areutilized. Example of cancer antigens include MAGE 1, MAGE 2, MAGE 3,tyrosinase, tyrosinase related protein 1 and 2, Pmel H. In the case ofviruses examples of antigens are proteins for example from HCV, HIV,HPV, HBV, influenza, rhinoviruses.

[0116] Ubiquitination Recognition Elements

[0117] In order to develop the compounds of the invention for targetedubiquitination, identification of a chemical element able to replace thetargeting and/or signaling activity of the N-terminal amino acid of aprotein or a sequence element of a protein, which result in theubiquitination of the protein, is required. A number of chemicalentities' ubiquitination recognition elements' have already beendescribed which interact with the ubiquitination system of the cell; anumber of di-peptides, and some modified amino acids. These compoundshave been described based on the ability to inhibit the activity of theubiquitination pathway based on the known activating amino acids fromthe N-end rule. These include dipeptides, amino acid hydroxamates, andamino acid methyl esters with small uncharged, basic or bulkyhydrophobic N-terminal residues (Gonda et al 1989, J Biol. Chem. 264:16700). In addition sequence elements have been defined which include,‘destruction box’ or D box, PEST motifs, Deg1, Deg 2, delta (δ) domains,and phosphorylated sequences which also target ubiquitination. Alsoconsidered as ubiquitination recognition elements are oxidizedderivatives of peptides. These compounds are useful in the presentinvention. Examples of oxidized amino acid are oxidized methionine toform methionine sulfoxide, oxidized leucine to form hydroxyleucine,oxidized tryptophan to form N-formyl-kynurenine, and oxidized tyrosineto form 3,4-dihydroxyphenylalanine.

[0118] It is also understood that identification of new ubiquitinationrecognition elements is possible using standard methods for drugdiscovery (as outlined below) based on modulation of the ubiquitinationpathways. Methods of screening compounds which can be used asubiquitination recognition elements has been demonstrated fordipeptides, amino acid hydroxamates, and amino acid methyl esters withsmall uncharged, basic or bulky hydrophobic N-terminal residues and alsolarge chemical libraries (U.S. Pat. No. 5,766,927; WO 98/23283; GB2,320,570 Gonda et al 1989, J Biol. Chem. 264: 16700). In one specificexample a compound was identified ‘compound of example 2’(1-chloro-2,4-bis{4-[2-chloro-6-(5-(2,7-disulfo-4-hydroxy-3-(2-(1-sulfonaphthyl)azo)naphthyl)amino)-1,3,5-triazinyl]amino}benzene),of patent application GB 2,320,570 (which is hereby incorporated byreference in its entirety), which inhibited an E2 ubiquitinationreaction indicating its utility as a ubiquitination recognition elementof the subject invention; here in called compound Z. Other equivalentmethods for identification of chemical elements equivalent to the PEST,Deg and other sequence elements based on the assays for ubiquitinationsystems (Hochstrasser M and Varshavsky A 1990, Cell 61; 697-708) can beused. In the case of the PEST sequence from ornithine decarboxylase(amino acids 422-462), this sequence has been fused to a greenfluorescent protein sequence to generate a fluorescent protein which hasa half life of only 2 hours from its original>24hours making this anideal system in which to detect molecular equivalent of the PESTsequence. In addition to specific molecular species which interact withthe E3 elements of the ubiquitination pathway, it is also contemplatedthat specific molecular species that interact with the E2 elements ofthe ubiquitination pathway can be used in an equivalent way to targetselective ubiquitination. A specific example of an E2 domain that isinvolved in targeting specific ubiquitination is the C-terminal domainsof E2. Thus chemical elements able to bind to the E2's and especiallythe C-terminal domains of E2 are considered ubiquitination recognitionelements of the subject invention. The elements that interact with theE2 elements have been defined as various sequence elements (parts) ofproteins that control their ubiquitination.

[0119] Analysis of the E3s has determined common themes in structure andfunction. The basic function for E3s is the recognition of a proteinsubstrate for ubiquitination. This is achieved either as a singleprotein or as a multi-protein complex. In some cases the E3s are singleproteins which typically depend on E2 to mediate the ubiquitination. Inthe case of one class of E3s known as SCF complexes (containing Skp1p,Cdc53p and F-box proteins in a complex) it is known that the F-boxproteins act as the substrate specific adapters to recruit varioussubstrates to the complex for ubiquitination. Thus in these E3s it isthe interaction of the F-box proteins with the proteins targeted forubiquitination, the ubiquitination is achieved through Cdc-34p (E2). Theabove description of the ubiquitination elements has drawn on generalnames for the elements such as E1, E2 and E3 but also some specificnames of the proteins in a given system. It will be understood by thoseskilled in the art that equivalent proteins, as determined by functionand sequence homology exist and can be considered to be equivalent(Patton E E, et, al. Trends Genet, 1998 14, 236-243).

[0120] Thus it is clear to those skilled in the art that bindingmolecules which bind to the ubiquitination recognition site (FIG. 1) ofthe ubiquitination system can be selected and identified using art knownmethods and available chemical libraries. In the absence of availablechemical libraries, synthesis of equivalent chemical libraries can bedone following art known methods, as described below for the discoveryof ubiquitination recognition elements of the subject invention.

[0121] Examples of PEST sequences include, MEFMHISPPEPESEEEEEHS (SEQ IDNO 1), MEFMHESHSS (SEQ ID NO 2), MEFMHISPPEPESHSS (SEQ ID NO 3),MEFMHESEEEEEHSS (SEQ ID NO 4), MEASEEEEEF (SEQ ID NO 5),HGFPPEVEEQDDGTLPMSCAQESGMDRH (SEQ ID NO 6), HGFPPAVAAQDDGTLPMSCAQESGMDRH(SEQ ID NO 7), HGFPPEVEEQDDGALPMSCAQESGMDRH (SEQ ID NO 8),HGFPPEVEEQDDGTLPMSCAQESGMDHH (SEQ ID NO 9), HGFPPEVEEQDVGTLPMSCAQESGMDRH(SEQ ID NO 10), HGFPPEVEEQDVGTLPISCAQESGMDRH (SEQ ID NO 11),HGFPPEVEEQDASTLPVSCAWESGMKRH (SEQ ID NO 12), FPPGVEEPDVGPLPVSCAWESGMKRH(SEQ ID NO 13), FLAEVEEQDVASLPLSCACESGIEYPA (SEQ ID NO 14),

[0122] FXXEVEEQDXXXLPXSCAXESGXX(X) (SEQ ID NO 15),FXXAVAAQDXXXLPXSCAXESGXX(X)X (SEQ ID NO 16), orHGXXPEVX(XX)DXXXLXXSCAQESGMXXX (SEQ ID NO 17),

[0123] where X is any amino acid and (X) is an optional amino acid.

[0124] Examples of D boxes include, RHALDDVSN (SEQ ID NO 18), RLALNNVTN(SEQ ID NO 19), RAALGDVSN (SEQ ID NO 20), RQVLGDIGN (SEQ ID NO 21),RAALGDLQN (SEQ ID NO 22), RAALGNISN (SEQ ID NO 23), RNTLGDIGN (SEQ ID NO24), RTALGDIGN (SEQ ID NO 25), RAALGEIGN (SEQ ID NO 26), RAVLEEIGN (SEQID NO 27), RSAFGDITN (SEQ ID NO 28), RSILGVIQS (SEQ ID NO 29), RAALGVITN(SEQ ID NO 30), RTVLGVIGDN (SEQ ID NO 31), RTVGVLQEN (SEQ ID NO 32),RAALGTVGE (SEQ ID NO 33), RTVLGVLTEN (SEQ ID NO 34), RAALAVLKSGN (SEQ IDNO 35), RLPLAAKDN (SEQ ID NO 36), RQLFPIPLN (SEQ ID NO 37), RRTLKVIQP(SEQ ID NO 38),

[0125] expressed as a general structure R(A/T)(A)LGX(I/V)(G/T)(N) (SEQID NO 39), or expressed as a consensus RXXLGXIXN (SEQ ID NO 40), where Xis any amino acid and amino acids in parentheses occur in more than 50%of known destruction sequences.

[0126] Examples of other ubiquitination recognition elements are;KEFAVPNETSDSGFISGPQSS (cactus) (SEQ ID NO 40), KGPDEAEESQYDSGLESLRSLR(IkBepsilon) (SEQ ID NO 41), KAADADEWCDSGLGSLGPDA (IkBbeta), (SEQ ID NO42), KKERLLDDRHDSGLDSMKDEE (IkBa1pha), (SEQ ID NO 43),

[0127] KX(8-10)DSG(hydrophobic amino acid)XS (SEQ ID NO 44), where the Sin bold are phosphorylated. In addition to the signals associated withNF kB activation are the related ubiquitination recognition elementsSYLDSGIHSGAT (SEQ ID NO 45), (human beta-catenin) and RAEDSGNESEGE (SEQID NO 46), (HIV-1 Vpu) where the S in bold are phosphorylated.

[0128] The identified and/or discovered chemical entities which bind tothe sites on the E3 and/or E2 elements involved in recognition prior toubiquitination are the ubiquitination recognition elements of thesubject invention. The ubiquitination recognition elements are thusfunctionally defined by their ability to compete for binding of thenatural recognition signals for ubiquitination with their ubiquitinationpartners, have a molecular weight less than 30,000; 50 to 10,000; 50 and3,000; 100 and 3,000; 200 and 3,000, are capable of being linked toother molecular species and retain their ability to compete for bindingof the natural recognition signals for ubiquitination with theirubiquitination partners. In addition the binding affinity of theseubiquitination recognition elements is typically greater than 10² M⁻¹.The binding affinity in a advantageous embodiment is greater than 10³M⁻¹. The binding affinity the most advantageous embodiment is greaterthan 10⁴ M⁻¹.

[0129] Some examples of ubiquitination recognition elements based on theN-recognin include;

[0130] Arg-εAhx-Cys

[0131] Arg-β-Ala-εAhx-Cys

[0132] Arg-εAhx-εAhx-Cys

[0133] Phe-εAhx-Cys

[0134] Phe-β-Ala-εAhx-Cys

[0135] Phe-εAhx-εAhx-Cys

[0136] Arg-Ala-εAhx-Cys

[0137] Arg-Ala-β-Ala-εAhx-Cys

[0138] Arg-Ala-εAhx-εAhx-Cys

[0139] Phe-Ala-εAhx-Cys

[0140] Phe-Ala-β-Ala-εAhx-Cys

[0141] Phe-Ala-εAhx-εAhx-Cys

[0142] Ubiquitination Recognition Peptide Element

[0143] Ubiquitination recognition peptide element, is defined as apeptide based moiety which is able to bind with a ubiquitination systemsproteins (other than those of the N-end rule) or its component proteins.This binding is further defined by the ability of the peptide moiety topromote the ubiquitination of a protein attached directly or indirectlyto the moiety.

[0144] Examples of such ubiquitination recognition peptide elements are;MEFMHISPPEPESEEEEEHS (SEQ ID NO 1), MEFMHESHSS (SEQ ID NO 2),MEFMHISPPEPESHSS (SEQ ID NO 3), MEFMHESEEEEEHSS (SEQ ID NO 4),MEASEEEEEF (SEQ ID NO 5), HGFPPEVEEQDDGTLPMSCAQESGMDRH (SEQ ID NO 6),HGFPPAVAAQDDGTLPMSCAQESGMDRH (SEQ ID NO 7), HGFPPEVEEQDDGALPMSCAQESGMDRH(SEQ ID NO 8), HGFPPEVEEQDDGTLPMSCAQESGMDHH (SEQ ID NO 9),HGFPPEVEEQDVGTLPMSCAQESGMDRH (SEQ ID NO 10),HGFPPEVEEQDVGTLPISCAQESGMDRH (SEQ ID NO 11),HGFPPEVEEQDASTLPVSCAWESGMKRH (SEQ ID NO 12), FPPGVEEPDVGPLPVSCAWESGMKRH(SEQ ID NO 13), FLAEVEEQDVASLPLSCACESGIEYPA (SEQ ID NO 14),

[0145] FXXEVEEQDXXXLPXSCAXESGXX(X) (SEQ ID NO 15),

[0146] FXXAVAAQDXXXLPXSCAXESGXX(X)X (SEQ ID NO 16), orHGXXPEVX(XX)DXXXLXXSCAQESGMXXX (SEQ ID NO 17), where X is any amino acidand (X) is an optional amino acid.

[0147] Examples of D boxes include, RHALDDVSN (SEQ D NO 18), RLALNNVTN(SEQ ID NO 19), RAALGDVSN (SEQ ID NO 20), RQVLGDIGN (SEQ ID NO 21),RAALGDLQN (SEQ ID NO 22), RAALGNISN (SEQ ID NO 23), RNTLGDIGN (SEQ ID NO24), RTALGDIGN (SEQ ID NO 25), RAALGEIGN (SEQ ID NO 26), RAVLEEIGN (SEQID NO 27), RSAFGDITN (SEQ ID NO 28), RSILGVIQS (SEQ ID NO 29), RAALGVITN(SEQ ID NO 30), RTVLGVIGDN (SEQ ID NO 31), RTVGVLQEN (SEQ ID NO 32),RAALGTVGE (SEQ ID NO 33), RTVLGVLTEN (SEQ ID NO 34), RAALAVLKSGN (SEQ IDNO 35), RLPLAAKDN (SEQ ID NO 36), RQLFPIPLN (SEQ ID NO 37), RRTLKVIQP(SEQ ID NO 38),

[0148] expressed as a general structure R(A/T)(A)LGX(I/V)(G/T)(N) (SEQID NO 39), or expressed as a consensus RXXLGXIXN (SEQ ID NO 40), where Xis any amino acid and amino acids in parentheses occur in more than 50%of known destruction sequences.

[0149] Examples of other ubiquitination recognition elements are;KEFAVPNETSDSGFISGPQSS (cactus) (SEQ ID NO 40), KGPDEAEESQYDSGLESLRSLR(IkBepsilon) (SEQ ID NO 41), KAADADEWCDSGLGSLGPDA (IkBbeta), (SEQ ID NO42), KKERLLDDRHDSGLDSMKDEE (IkBalpha), (SEQ ID NO 43),

[0150] KX(8-10)DSG(hydrophobic amino acid)XS (SEQ ID NO 44), where the Sin bold are phosphorylated. In addition to the signals associated withNF kB activation are the related ubiquitination recognition elementsSYLDSGIHSGAT (SEQ ID NO 45), (human beta-catenin) and RAEDSGNESEGE (SEQID NO 46), (HIV-1 Vpu) where the S in bold are phosphorylated.

[0151] Ubiquitination Recognition Signal

[0152] Ubiquitination recognition signal is a sequence of a proteinwhich is known to act as the signal for ubiquitination systems. Thisubiquitination recognition signal has the ability to promote theubiquitination of a protein attached directly or indirectly to thesignal.

[0153] Method for the Selection of the Target Protein Binding Elements

[0154] The subject invention provides a significant advantage over theexisting art as it makes use of binding to develop drugs and othercompounds with activity against selected target proteins. This advanceover the traditional methods is that the invention obviates the need tofind a compound which binds to a specific site, by making the wholeprotein surface available for the development of drugs and otherbiologically active compounds. This approach thus provides a new avenuefor the discovery and selection of novel pharmaceuticals, drugs andother valuable biologically active compounds.

[0155] It is understood by those skilled in the art that methods for thediscovery of target protein binding elements to a pre-selected (target)proteins (targets of the subject invention) are well know. Examples arereferenced as follows, Karet G, Drug Discovery and Development. January1999, 32-38, www.rdmad.com/drug; Bohm. H-J and Klebe, G., 1996, Angew.Chem. Int. Ed. Engl. 35, 2588-2614; Angew Chem Int Ed Engl 1996, 35,2288-2337; Bunin B A., 1996, Methods in Enzymology, 267, 448-465; PatekM. 1995, Tetrahedron Let., 36, 2227-2230; Nestler H P., 1996, Bioorg.Med. Chem. Lett., 6(12), 1327-1330; Look, G C., 1996, Bioorg. Med. Chem.Lett., 6(6), 707-712; Nakayama G R., 1998, Curr. Opin. Drug Discoveryand Development 1(1), 85-91; Hill D C., 1998 Curr. Opin. Drug Discoveryand Development 1(1). 92-97; Bright, C., 1998, Bioorganic and Med. Chem.Lett. 8, 771-774; Forbes I T., 1998, J Med. Chem. 41(5), 655-657; whichare hereby incorporated by reference in their entirety.

[0156] The binding molecules of the subject invention are defined bybinding to the selected target molecule, having a molecular weight lessthan 30,000; 50 to 10,000; 50 to 3,000; 50 to 1,000; 100 to 3,000; 200to 3,000 and 300 to 3,000. Also the binding molecule is defined bv thebinding affinity which is typically greater than 10⁵ M⁻¹. The bindingaffinity in an advantageous embodiment is greater than 10⁶ M⁻¹. Thebinding affinity in a more advantageous embodiment is greater than 10⁷M⁻¹. The binding affinity in the most advantageous embodiment is greaterthan 10⁸ M⁻¹.

[0157] Large libraries of compounds exist in numerous places. Thesecomprise compounds isolated from various natural sources in addition tothose generated denovo or partially denovo from natural precursororganic molecules. The sources for various compound libraries include:ArQule (www.arqule.com); Pharmacopeia (www.pharmacopiea); Cerep(www.cerep.com); Merk; Glaxo-Welcome; Zenova; Sigma-Aldrich; OxfordAsymmetry International (www.oai.co.uk); Specs and BioSpecs(www.specs.net); AsInEx (www.asinex.com); ComGenex, Princeton, N.J.;Panax, New York, N.Y.;

[0158] Synthetic Approaches for Compound Generation to Screen for TargetProtein Binding Elements and Ubiquitination Recognition Elements of theSubject Invention

[0159] Combinatorial chemistry has been widely adopted by large andsmall drug discovery companies alike since 1990. This is a set oftechniques for creating a multiplicity of compounds and then testingthem for activity (Angew Chem Int Ed Engl 1996, 35, 2288-2337).Combinatorial chemistry is used to generate large libraries of moleculesinstead of synthesizing compounds one by one, as has been donetraditionally. These libraries are screened using high-throughputscreening to identify the most promising pharmaceutical compounds.Typical rates for success are around 0.1% and libraries of around200,000 compounds are typically screened. These initial hits in screensare then further analyzed for other desired drug properties for exampledrug metabolism, bio-availability, stability, potency, and cost. Thus,the discovery of compounds with binding and inhibitory activity is aroutine practice. This is especially true if only binding is screenedfor independent of modulation to the target's activity of interest.

[0160] Combinatorial chemistry was first conceived in 1984. Initially,the field focused primarily on the synthesis of peptide andoligonucleotide libraries. In 1984 H. Mario Geysen and his groupdeveloped, a technique for synthesizing peptides on pin-shaped solidsupports. In 1985, Richard A. Houghten, developed a technique in whichtiny mesh packets, act as reaction chambers and filtration devices forsolid-phase parallel peptide synthesis.

[0161] The field's original predominant focus on peptide andoligonucleotide libraries began to change about 1991 with thedevelopment of combinatorial techniques for producing small organicmolecules with molecular weights of about 1,000; a class of compounds inwhich drugs and other valuable bioactive small molecules are most oftenfound.

[0162] Two basic methods are used in combinatorial chemistry solid-phaseand solution-phase methods. Using these methods combinatorial compoundsare created either by solution-phase synthesis or by producing compoundsbound covalently to solid-phase particles.

[0163] Solid Phase Methods

[0164] Solid Phase Methods: (Fruchtel J S., and Jung G., 1996, Angew.Chem. Int. Ed. Engl. 35, 17-42; which is incorporated by reference inits entirety).

[0165] Solid-phase synthesis makes it easier to conduct multistepreactions and to drive reactions to completion, because excess reagentscan be added and then easily washed away after each reaction step.Another key factor in favor of solid-phase synthesis is that it makes itpossible to use split synthesis, a technique developed in 1982. Splitsynthesis produces large support-bound libraries in which eachsolid-phase particle holds a single compound, or soluble librariesproduced by cleavage of compounds from the solid support. For example ina split synthesis method if you have 3 compound addition steps with 10compounds used at each step i.e. 10 containers for those compounds. Thiswill generate 10³ compounds. Also if you consider all the reaction stepsincluded in a synthesis 10,000 compounds made via a solid phase methodsusing a three-step chemistry may only require about 22 containers forthe chemistry and about 66 liquid handling steps relative to the 10,000containers and 30,000 liquid handling steps. When you combine theseadvantages of solid phase synthesis with split synthesis a significantlevel of synergy is achieved.

[0166] A potential disadvantage of solid-phase synthesis is that ahydroxyl, amine, carboxyl, or other polar group are typically present ona molecule to be able to attach it to a solid support. This is apotentially undesirable constraint on the structure of compoundssynthesized on solid phase, because products retain the polar group evenafter they are cleaved from the support. Several groups, have devisedtraceless linkers that avoid this problem, because the linkers areremoved completely from products during the cleavage process. Forexample an acylsulfonamide linker that can be displaced by variousnucleophiles to add diversity to a library has been described. Anotheralternative to the traceless linker has been developed using achemistry, in which reagents used to cleave products from the solidsupport are incorporated into the product. Using this method a singlecompound on a solid phase can give rise to a chemical series based onthe reagents used to release the products. This can be achieved via theuse of substoichiometric amounts of different cleaving reagents,sequentially reacted with a compound that is synthesized on areact-and-release type resin, and each product is individually eluted.This protocol has advantages when combined with automated chemistrysystems such as those used for peptide and oligonucleotides synthesis.The number of compounds generated with this method can be up to 10 timesthe number of chemistries generated on the solid phase. The result isalso a relatively pure product in solution. This method is an example ofthe combinations of solid and solution phase chemistries.

[0167] In order to solve one of the problems caused by the use of splitsynthesis methods, namely knowing which compound in the library showsactivity, encoded libraries have been constructed. An example of anencoding technique is one based on inert halogenated compounds that areused to record the chemical reaction history of each support bead. Thetags can be analyzed by capillary gas chromatography with electroncapture detectors and autosamplers to rapidly reveal the identity ofactive compounds in the library. In addition to this method compoundscan be released from the bead and analyzed by MS and/or GC/MS. Otheralternatives to the deconvolution of the library are based on theresynthesis of sub set pools from a positive hit of pooled compounds.One exciting approach to this problem of compound identification (from ascreen of pooled compounds), is based on the use of affinity selectionplus size exclusion chromatography (to separate bound compounds fromthose that have little or no affinity for the target protein), followedby mass spectroscopy, to identify leads that bind to the target ofinterest. This method eliminates the need to encode the library andmakes use of the molecular weight of the compound as the tag. Someproblems may be encountered from redundancy of some molecular weightswithin a library, but higher resolution and fragmentation MS methods canbe used effectively. In addition combinations of these approaches can beconsidered where the tag is left attached to the compounds which bind tothe target molecule of interest and are then selectively eluted andsubjected to cleavage releasing the tag or code which can then beidentified by MS or GC/MS methods (Karet G, Drug Discovery andDevelopment, January 1999, 32-38, www.rdmad.com/drug; which is here byincorporated by reference in its entirety)

[0168] Solution Phase Methods

[0169] Solution phase chemistry is favored by many for libraryconstruction due to the wider range of organic reactions available forsolution-phase synthesis, the technology used traditionally by mostsynthetic organic chemists, and products in solution can be more easilyidentified in standard drug target assays and characterized. A problemfor solution-phase synthesis of one molecule at a time is the finalpurification that can be both expensive and slow. Chromatography iscommonly a first resort since it usually works. In addition, theproblems associated with solution chemistry are compounded whenattempting to make tens of thousands of compounds to generate a libraryor a ‘book’ for a library.

[0170] In the generation of libraries of chemistries numerous methodshave been devised resulting in the wide spread use of large libraries ofchemicals to readily allow the discovery of potential drug candidates.The generation of chemical libraries that are free in solution istypically the goal of most of the pharmaceutical industry. This aim isdue to the nature of many of the drug targets and the associated assays.Also the construction and utility of chemical libraries is typicallyfacilitated but the generation of master plates of compounds in solutionto form the basis of the chemical library. Thus the general advantagesof the solid phase synthesis methods are typically not fully realized inthe context of the current drug discovery efforts. The main reason forthis is the interest not in binding of the compound to the drug targetbut to demonstrate that the activity of the drug target is altered,which typically requires compound free in solution. Further concernswith libraries of compounds on a solid phase arise from concerns of thepotential influence of the linker and steric effects on the compoundsbound to the solid phase.

[0171] Thus methods for the discovery of compounds which bind to targetmolecules is known in the art. Also, the optimization of the initiallydiscovered compound is well known in the art where the affinity isimproved by generation of a pool of related compound via a moreselective combinatorial chemistry approach.

[0172] The present invention provides a mechanism to overcome theseproblems in drug and small molecule discovery.

[0173] Embodiments of the Subject Invention

[0174] Compounds Active on 5-Lipoxygenase as Anti-asthmatics

[0175] Screening for Target Protein Bindings Elements

[0176] Initially a target protein is selected, for example5-lipoxygenase which is a molecule involved in inflammatory reactionsespecially in asthma. Target protein for the subject invention come fromnumerous fields where small molecules are used to achieve modulation ofa biological system in eukaryotic organisms. Examples of such fields areinsecticides, fungicides, antivirals, herbicides, anti-parasitics andherbicides when applied to humans, animals and plants.

[0177] The target protein is then either purified from a natural sourcein order to provide sufficient material for the screen or expressed viarecombinant methods to provide sufficient material for the screens.

[0178] The target protein is then either labeled directly with adetectable species such as a radioactive, electrochemiluminescent, andchemiluminescent or fluorescent label or with an indirectly detectablespecies such as an enzyme, or particle. Alternatively an antibody orequivalent with binding activity to the 5-lipoxygenase is labeled.

[0179] The next step is to buy a library of compounds for screening. Alibrary of from 1,000 to 1,000,000 is typical of the size that isscreened. These are available from a series of companies as describedearlier. These libraries of compounds are used to screen for the bindingof the target protein 5-lipoxygenase. Ideally compounds are bought stillbound to the solid phase or are screened for binding directly toimmobilized target protein 5-lipoxygenase using methods as describedbelow for screening.

[0180] It is also possible to generate a chemical library of variouspotential binding molecules bound to a solid phase followingconventional methods to give rise to differing potential compounds. Theoptimal methods for the construction of the chemical library is toemploy the methods of split synthesis coupled to the solid phase (asoutlined above). The library is generated using a series of solid phasechemistries such as to give rise to various ‘chemical books’ that incompilation form the basis of a library. The library is screened in theform of a library or in the form of the ‘chemical books’. Typically onewould take the products from the split synthesis and pool the solidphase and use this as the basis for the screen.

[0181] To the pool of beads used as the solid phase for the synthesis, amixture of buffer, detergents, salts and blocking agents such as serumalbumin or other proteins are added. This buffer addition step is usedto ‘block’ the beads or solid phase in such a way that any significantnon specific binding of the selected target (5-lipoxygenase) does notoccur. Following this blocking step the beads are washed and followed bythe addition of the 5-lipoxygenase either labeled or not. The beads orsolid phase are then incubated to allow the binding of the targetprotein binding elements to the target, in this case 5-lipoxygenase.Following the incubation of the target molecule to the beads or solidphase the beads are washed and then the binding of the labeled5-lipoxygenase detected directly. In an alternative format, if the5-lipoxygenase is labeled with an indirectly detectable label such as anenzyme, the beads are then placed in to a substrate reaction solution todetect the presence of the enzyme label. In the case of an enzyme label,substrates for these detection methods are based on insolublechromogenic products. In the case where the 5-lipoxygenase is notlabeled and an antibody or equivalent is available, the beads aresubjected to another binding reaction where the antibody or equivalent,is labeled either directly or indirectly as suggested for the labelingof 5-lipoxygenase. It is also possible at this step to not use a labeledantibody or equivalent and to add a further step where the labeledantibody or equivalent is used. These additional steps can be detectedusing the same standard methods known in the art as suggested for thedirectly labeled 5-lipoxygenase.

[0182] Following these steps a series of beads are identified and thesebeads are selected from the bead population and subject to analysis todetermine the structure of the binding molecule that is able to bind the5-lipoxygenase as in this example. This is achieved by the use of GC/MSor via molecular tags used during the construction of the library asdescribed earlier. Alternatively a pool which was positive is re-madegenerating a series of sub pools for screening and further re-synthesisand dividing out of the various pooled compounds until a single compoundis presented in a single well for analysis allowing the determination ofthe active compound.

[0183] Addition of the Ubiquitination Recognition Element

[0184] At this point in the compound discovery path for the subjectinvention, the target protein-binding element of the compounds of theinvention has been identified. These optimal binding molecules are thensubjected to further chemistry to add the ubiquitination recognitionelement.

[0185] An alternative approach to the discovery of the targetprotein-binding element is based on solution phase screening. In such anexample compounds (available either via synthesis, natural products orfrom companies such as ArQule (www.arqule.com), Pharmacopeia(www.pharmacopiea), and Cerep (www.cerep.com) are obtained and added tothe target protein of interest and then subjected to size exclusion toremove the unbound compounds. The protein bound fraction is thensubjected to GC/MS to identify the molecules. In this way the solutionphase screening is made rapid and facile for compounds in solution.

[0186] Compounds Active on IL-4 Receptor as Anti-asthmatics

[0187] Introduction

[0188] A further embodiment of the subject invention is the developmentof a compound targeted to a receptor involved in development of asthma.In recent studies into the pathophysiology of asthma, IL-13 has beendemonstrated to be the central mediator acting through the IL-4receptor. Thus asthma can be controlled by the lowering of either theIL- 13 or IL-4 receptor. The IL-4 receptor consists of two subunits; a140 kd alpha subunit, which binds IL-4 or IL-13 and transduces theirgrowth-promoting and transcription activating functions and a gamma csubunit, common to several cytokine receptors, which amplifies signalingof IL-4 receptor alpha. In this application of the subject invention thetarget for drug development is the IL-4 receptor alpha chain. The IL-4receptor alpha chain has a large intra cellular protein domain thatforms the specific molecular target of the discovery approach of thesubject invention.

[0189] Expression of the IL-4 Receptor Alpha Chain

[0190] Initially the IL4 receptor alpha (IL-4a) chain intra-cellulardomain is cloned from human blood lymphocytes. The cloned DNA isengineered to generate a gene sequence that directs the expression ofthe cytoplasmic domain of the IL-4a chain. This gene sequence is alsoengineered to include a sequence tag that allows the purification anddetection of the expressed receptor sub-unit. This expression is carriedout using various methods known in the art. Methods for expression ofproteins, are numerous; an example of one is one of the vectors fromInvitrogen (Carlsbad, Calif.) such as the His-Patch ThioFusion, whichallows for the optimal expression of proteins in a soluble form andcontaining a His tag which allows rapid purification. This system allowsfor the production of soluble protein after cleavage using theenterokinase cleavage site in the cloning vector pThioHis A, B, C. Analternative Xpress system also provides a useful expression system whichallows rapid purification via a His sequence and also a proteasecleavage site to yield the protein of interest with out the Hissequences. One of the vectors from the Xpress system, pTrcHis2 A, B, Cseries is especially useful; this vector allows the use of the Hissequence for purification but also allows for the tagging of the proteinwith a myc epitope for detection and assays for the expressed proteincontaining the epitope tag sequence myc with an anti-myc antibody.Expression vectors are also supplied by other vendors such as NewEngland Biolabs (Beverly, Mass.) whose pMAL-c2 and pMAL-p2 vectorsprovide an expression system for E.coli which provides a tag which ismaltose binding protein (MBP), this tag can be used in purification andalso in detection of the fusion protein. The MBP can be removed by theuse of the factor Xa cleavage site.

[0191] Following the cloning of the IL-4a cytoplasmic domain, using artknown methods for the cloning and expression of proteins, therecombinant protein is expressed and purified using the tag sequenceattached during the cloning. This purified receptor sub-unit is thensubjected to screening against a chemical library.

[0192] Screening for Binding Molecules from Chemical Libraries

[0193] The step of screening for specific molecules is made easy in thisinvention as only binding activity is desired and not specificmodulation of the target protein as is required in traditional drugdiscovery.

[0194] The next step is to buy a library of compounds for screening. Alibrary of from 1,000 to 1,000,000 is typical of the size that might bescreened. These are available from a series of companies as describedearlier. These libraries of compounds are used to screen for the bindingof the target protein 5-lipoxygenase. Ideally compounds are bought stillbound to the solid phase or are screened for binding directly toimmobilized target protein 5-lipoxygenasen using methods as describedbelow for screening.

[0195] It is also possible to generate a library of from 1,000 to100,000 compounds contained on a solid phase using split synthesismethods as described earlier. This library is constructed using a seriesof chemical methods resulting in pools of the solid phase used duringsynthesis which form the basis of the ‘books’ which go to make up thelibrary. In addition at the final chemical coupling step used toconstruct the various books the solid phase pools are stored insub-pools forming ‘chapters’ of the ‘books’ in the libraries. These socalled ‘chapters’ form the basis for screening as they contain not onlypools of compounds but also a known chemical-coupling step used in thesynthesis of the ‘chapters’ of the library.

[0196] The library can then be screened using two approaches. In bothcases the solid phase from the chemical library to be screened issubjected incubation with assay buffers with blocking agents such as forexample; proteins (i.e. BSA, gelatin), polyvinylpyrrolidone, ficoll,heparin, detergents (i.e. SDS, Tween, NP40, Triton X- 100). Thisincubation step is to block the non-specific binding sites on the solidphase used in the generation of the library and allow the determinationof specific binding events. This initial incubation is an art recognizedstep in various binding assays such as ELISA, southerns, westerns etc.Following this incubation with blocking agents the protein of interestis then added to a buffer which typically has the same composition asthat during the blocking step but can also be modified using lower or noadditional blocking agents with the exception of the detergents whichare typically always present during a binding reaction.

[0197] In one of the screening methods the ‘chapters’ of the various‘books’ following the blocking step are then subjected to binding withthe purified receptor sub-unit. The solid phase from this incubation isthen washed and subjected to a second binding step with a labeledreagent which binds to the tag sequence added to the receptor sub-unitduring the recombinant engineering for the expression of the receptorsub-unit. Typically an antibody to this tag recognizes the tag sequence;examples that are in common use are the myc, flag, and his epitopes.Following the incubation with the tag specific binding species thepresence of the labeled binding species is detected by the presence ofthe label that is typically an enzyme such as alkaline phosphatase orperoxidase. The detection step typically makes use of an insolublechromogenic substrate that is readily detected by eye or by imageanalysis systems.

[0198] In an alternative method soluble substrates can also be used andscreened using ELISA plate readers, eye or other spectrophotometricmethods. In its simplest form the various ‘chapters’ of the ‘book’ fromthe library are screened by eye to look for beads that have developed acolor due to the enzymatic action on the chromogenic substrate. Thesecolored beads indicate that the receptor sub-unit is binding to one ofthe compounds within the ‘chapter’ the next step is to determine ifthese so called positive ‘chapters’ contain specific binding or ifbinding is just to the tag binding reagent or some non-specificactivation of the chromogenic substrate. To achieve this, the positive‘chapters’ are screened with out the specific binding step to thereceptor sub-unit. If these positive ‘chapters’ now become negative orshow significantly reduced signals interms of positive solid phases within the mixture then these are considered to be real positive hits in thescreen. These real positive ‘chapters’ are then subjected tore-synthesis. In this re-synthesis the initial chemical steps to createthe specific binding molecule is unknown only the last chemical couplingstep in the compound synthesis is know, as this formed the last chemicalstep which constructed the ‘chapter’. During the re-synthesis of thepositive chapter the chemical step prior to the last chemical couplingis carried out as in the initial synthesis but the solid phase is notpooled and split for the final chemical coupling but are maintained asseparate pools then subjected to the chemical coupling step know forthat chapter. This re-synthesis results in the formation of a new seriesof solid phase compound pools which have the last two chemical couplingsteps known. This new series of solid phase compound pools are screenedas in the initial screen and positive pools are checked as previouslyfor the binding specificity to identify positive pools. The positivepool(s) now allow the re-synthesis of the pool(s) with the last twosteps for the generation of the compound which specifically binds to thereceptor sub-unit. The positive pools are then subjected to the samecycle of re-synthesis and screening as just described but with the lasttwo chemical coupling steps know the pools are maintained individuallyprior to the last know step. In this way the synthesis of the specificcompound able to bind to the receptor sub-unit is deconvoluted from thechemical ‘library’ and identified.

[0199] In an alternative method the positive solid phase is removed fromthe screen and collected. These are then subjected to the cleavagereaction which removed the specific chemistry from the solid phasefollowed by the analysis of the various chemical species using GC toseparate the individual compounds followed by MS to determine themolecular weight. This information coupled with the synthesis methodsused is used to determine the compound identity. After the determinationof these various candidate specific binding molecules they are thenre-synthesized and subjected to the binding assay to check if these arethe specific compounds that resulted in the positive solid phases.

[0200] Addition of the Ubiquitination Recognition Element

[0201] This screening effort following methods and protocols known inthe art allows the identification of compounds that bind to the receptorsub-unit. These compounds then form the basis for the development ofcompounds of the invention. These compounds are then subjected tofurther chemistry based on the use of the linker group used in thedevelopment of the solid phase chemistry. To this linker group thevarious ubiquitination recognition chemistries are added. This finalstep of chemistry generates the compound of the invention. The compoundof the invention are then subject to analysis to determine which of thecompounds from the chemical library screen with which of theubiquitination recognition elements is able to function most effectivelyin the targeted ubiquitination and/or degradation. In the case where theubiquitination recognition chemistry is based on the N-end rule therabbit reticulocyte lysate forms the basis for the assay using therecombinant produced receptor sub-unit labeled with for example ¹²⁵I tofollow the fate of the protein. In addition the compounds of theinvention can be tested in a mammalian tissue culture system where thetarget protein either intact or as an engineered fragment is expressed.In such a mammalian tissue culture system the compounds effect on thetarget protein's level is determined by making use of the tag sequencewhich can be engineered into the recombinant expression of the targetprotein during the construction of the mammalian tissue culture testsystem. The tag sequence is used to determine the levels of the targetprotein during the incubation with the potential compounds screened andsynthesized as described above. This assay for the tag sequence can takethe form of a western blot or via an ELISA, for example. Other tagswhich are valuable to use are those based on the green fluorescentprotein, which allows the analysis of protein levels in living cellsand/or organisms.

[0202] The compounds that show the optimal activity in the test systemswill then form the basis for the next stage of drug development. In thisnext stage these selected compounds are subjected to the recognized drugdevelopment path. The drug development path determines the potentialvalue of the compounds by evaluating a series of factors includingbioavailability; toxicology, pharmacology and efficacy in animal modelsbefore the compounds are considered for human testing.

[0203] Development of Pesticides

[0204] An alternative embodiment of the subject invention is thedevelopment of pesticides. Pesticide is a general classification thatincludes insecticides, rodenticides, fungicides, herbicides, andfumigants. The aim of the pesticide is the destruction of some life formand as such selectivity is desirable. The methods that have beendescribed for the subject invention for development of active compoundsto the 5-lipoxygenase and IL-4 Ra also apply to the development ofpesticides. Pesticides are also compounds of the invention, which aretargeted to an important biochemical pathway in a pest that is requiredfor its survival or prolonged viability. A pest is an organism that hassome direct or indirect deleterious effect on mankind. The term pest iswidely used to cover any organism that has some direct or indirectdeleterious effect on mankind. Some examples of pests are aphids, moths,lice, fleas, locusts, mice, rats, weeds etc. In the development of apesticide the methods outlined above are followed with the exception ofthe target in the case of developing pesticides key biochemical pathwaysfor survival in the pest would form the basis of the molecular targetselected to screen for protein binding elements. In the case ofinsecticides examples of key biochemical pathways include, ecdysone20-monooxygenase, ion channel of the GABA gated chloride channel,acetylcholinesterase, voltage-sensitive sodium channel protein, calciumrelease channel, and chloride channels.

[0205] The subject invention is ideally suited to the optimaldevelopment of pesticides due to subject inventions ability to rapidlyscreen for specific interactions which can be developed into a highlyspecies specific compounds of the invention. Specificity is of primeimportance for the development of pesticides as the targeted pests areeither present in close proximity to mankind, or as in the case ofagriculture pests, the pests are targeted on food intended for mankindwhere toxic compound residues are unacceptable. For example, in the caseof pesticides that are targeted to kill aphids, the compound is ideallytargeted only to the aphids and has no effect on the beneficial insectssuch as ladybugs, and bees. The subject invention provides through itsmolecular basis of compound selection, both a facile and an improvedmethod for the development of pesticides. In the development of anoptimal pesticide the specific target protein involved in the keybiochemical pathway is cloned and engineered using well known methods togenerate a source of protein for screening the chemical compound librarywhich has sequence tags to enhance the screening and characterization ofthe compounds of the invention. This process is also repeated using theproteins from the organisms also posses the same critical biochemicalpathways but are not the target pests. Thus a set of proteins can bedeveloped from the pest organism and from organisms which are likely tobe exposed to the compounds of the invention when used as pesticides. Inthe screening procedures as described earlier for the development of theanti-asthmatic compounds in addition to the screen for binding to thedesired target, absence of binding can also be screened for using theproteins from the non-pest organisms. In this way a set of compounds canbe selected which show specificity to the target pest organism and notcommonly encountered or related non-pest organisms. This type ofscreening is an advantage of the subject invention as it is based on theuse binding and does not require a complex activity assay. In this waythe subject invention provides for a low cost and rapid route to theselection of molecular species which have a high degree of speciesspecificity.

[0206] The subject invention allows for the development of selectivepesticides. The development of pesticides follows many of the previouslydescribed methods. The screen methods for the binding molecules thatrecognize the specific target have been described earlier for the IL-4receptor alpha chain and 5-lipoxygenase. These screens are used in orderto find molecules with the binding affinity for the target proteins ofinterest; for example to develop a specific herbicide the proteinenolpyruvylshikimate-phosphate synthase represents a good target as thisis the molecular target for glyphosate. The target protein is eitherpurified from the natural source or cloned and expressed using variousrecombinant methods to produce the enolpyruvylshikimate-phosphatesynthase. An ideal target for the development of a selective herbicideis poison ivy, in this case the enolpyruvylshikimate-phosphate synthasefrom poison ivy is used as the source of the target protein. The screenfor the binding molecules is initially focused on this target butsecondary screens are carried out on the various other plantsenolpyruvylshikimate-phosphate synthase normally present in the sameenvironment as poison ivy is found growing naturally. The secondaryscreen is used to establish the binding molecules that do not bind tothe other plant enolpyruvylshikimate-phosphate synthases in order toprovide the level of specificity desired. Following the identificationof the selective binding molecule this is then coupled to theubiquitination recognition element in order to generate the herbicide ofthe subject invention, which is selective to poison ivy. The developmentof the selectivity is further enhanced using comparative sequencing ofthe various molecular targets thus defining the various sequenceelements that are unique to poison ivy (or other target organism). Thesequence information then allows both the definition of the molecularbinding site within the molecule but also the sequences of the variousproteins that are used in the secondary screens to define thespecificity of the final binding molecules from the screen. It will beunderstood that the methods described in the subject invention benefitgreatly from the recent advances in genomic sequencing which make muchof the sequence information readily available or easily obtainable. Itwill be understood by those skilled in the art that these methods can beapplied readily to any pest in the development of pesticides. In thecase where no molecular target is known or can be defined it will beunderstood that the subject invention also allows a route to discoveryof such molecular targets. This discovery can be achieved through theselection of targets by various levels of homology with know targets orvia selection based on no homology which leads more rapidly to thedevelopment of selectivity even if it takes longer to define the roleand value of these new molecular targets. Other examples of targets forthe development of herbicides include, Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

[0207] Development of Compounds Effective Against Parasites

[0208] In the development of compounds that are targeted to parasiticorganisms the current invention provides for significant advantages. Ithas been traditionally a problem developing drugs that provide for theselective toxicity to various parasites of mankind and his domesticanimals. This problem has been largely due to the problems of culturingthese organisms and to the problems of finding a toxin that has thedesired level of toxicity to the parasite with out damaging the hostorganism. This problem has presented itself due to the large number ofrelated biochemical pathways that are shared between the eukaryoticorganisms. Some efforts have been made with some success to definebiochemical differences this has not yielded a broad range of targetsfor the development of drugs. The subject invention provides for a morefacile and optimal method for the development of compounds effectiveagainst parasites in the groups of Protozoan parasites: Balantidium,Cryptosporidium spp., Giardia spp, Plasmodia, Trypanosoma, Leishmania,Trichomonas, Entamoeba, Eimeria, Toxoplasma, Plasmodium, Babesia,Theileria Metazoan parasites: Nematode parasites, Ascaris spp.,Capillaria spp., Dracunclus spp., Enterobius spp., Filariasis due tovarious organisms, hookworm infections, Strongyloides spp., Toxocaraspp., Trichinella spp., Trichuris spp., Taenia spp., Diphyllobothriumspp., Hymenolepis spp., Echinococcus spp., Shistosoma spp., Fasciolopsisspp., Heterophyes spp., Metagonimus spp., Clonorchis spp., Opisthorchisspp., Paragonimus spp. etc.

[0209] Targets for compound development: Leishmania, proteins of thesterol synthesis pathway: Plasmodium, dihydrofolate reductase;dihydrofolate reductase-thymidylate synthase (bifunctional) resistanceknown due to mutations in the gene for this enzyme, heme polymerase:Trypanosoma, ornithine decarboxylase, trypanothione reductase, Ornithinedecarboxylase of the trypanosoma represents an desirable candidate fordestruction due to its long half-life and low turn over in trypanosoma.

[0210] Protein Level Control

[0211] This invention is also to a method for the control of proteinlevels with a cell. This is based on the use of compounds of theinvention which are known to interact with a specific protein or proteinsequence element. These specific proteins known to interact withcompounds of the invention are used to generate chimeric fusion proteinswith a desired target protein. These chimeric fusion proteins thusfunctionally link the ability to be destabilized by the compounds of theinventions to the desired target protein. In this way known compounds ofthe invention and known proteins and/or protein sequence elements can becombined to target the genetic engineering of another protein to renderit degradable and thus controllable by a compound of the invention. Thefollowing are by way of illustration of some possible application ofthis idea.

[0212] Control of Protein Levels Within a Cell

[0213] In another embodiment of the subject invention, control ofspecific gene products is achieved. In this embodiment a gene(s) isengineered such that its expression results in the production of thedesired protein but with the addition of a protein which has a specificbinding affinity for a small molecule. Examples of such sequences arestreptavidin, avidin, antibodies, single chain antibodies, thioredoxin,maltose binding protein, and the peptide motif CCXXCC (SEQ ID NO 47),and WEAAAREACCRECCARA (SEQ ID NO 48), (Griffin B A, 1998, Science 218,269). In the case of thioredoxin and the peptide motif CCXXCC (SEQ ID NO47), WEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA (SEQ ID NO49), these are known to bind to tightly to organoarsenical compounds.One potential binding species for the peptide motif CCXXCC andWEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA (SEQ ID NO 49),is 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein with otherbis-organoarsenical being useful (Griffin B A, 1998, Science 218, 269,which is hereby incorporated by reference in its entirety).

[0214] Having generated the modified gene for the protein of interestthese genes are then introduced into the cells desired either formingthe basis of a cell culture study in vitro or through the generation ofa transgenic animal which expressed the modified gene in its normalcontext or aberrantly to determine its role within the intact organism.An example of this type of engineering is described in Griffin B A,1998, Science 218, 269.

[0215] In this embodiment the compound of the invention is built aroundthe small molecule with a specific binding affinity for a specific aminoacid as exampled above. In the above example these are biotin bindingwith streptavidin and avidin, any small molecules binding with singlechain antibodies such as biotin, digoxin, fluorescein and theorganoarsenical compounds such as4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein, p-aminophenylarsineoxide binding with thioredoxin and the peptide motif CCXXCC (SEQ ID NO47), WEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA(SEQ ID NO49). To these binding molecules are attached the ubiquitinationrecognition elements to generate a bifunctional molecule which is ableto bind to the genetically engineered protein and activate theubiquitination of the engineered protein. Having generated thesebifunctional molecules these then are used to treat the cells and/ororganisms which contain the engineered protein. This treatment resultsin the rapid degradation of the engineered protein in a dose dependentfashion allowing the determination of the role of the various proteinsin the biology and/or physiology of the cell and/or organism. Thisembodiment of the subject invention allows the rapid generation of aseries of mutant proteins, making use of an identical compound andtreatment schedule in affecting changes within a cell and/or an organismthat allows for optimal determination of the role of various proteins inan controlled study. This is achieved with less perturbation of the celland/or organisms natural biochemistry than is possible with othermethods.

Control of Green Fluorescent Protein Levels

[0216] An example of the above embodiment is directed to thedemonstration of targeted ubiquitination to mediate degradation of aprotein inside living cells. The green fluorescent protein (GFP) ECFPplasmid vector (Clontech, Palo Alto, Calif.) was chosen in order toengineer the following binding site AEAAAREACCRECCARA (SEQ ID NO 49),into the C terminus of the expressed ECFP (GFP) following establishedmethods to form an expression vector able to direct the expression a GFPwith a C terminal tagged end ECFP-Cys4 (Griffin B A, et al 1998 Science,281, 269-272). This choice of the ECFP was also made so that theformation of the complex of4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein with the ECFP-Cys4demonstrates fluorescent energy transfer (FRET) from ECFP-Cys4 to thebound 4′,5′-bis(1 ,3,2-dithioarsolan-2-yl)fluorescein. This vector withthe ECFP-Cys4 gene is then transfected into HeLa cells and demonstratesthat expression is obtained and the protein had the expected long halflife of>20 hrs. Various compounds of the invention are made as follows;with 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein coupled using EDCchemistry, to a ubiquitin recognition elements selected fromArg-εAhx-Lys; Arg-β-Ala-εAhx-Lys; Arg-εAhx-εAhx-Lys; Phe-εAhx-Lys;Phe-β-Ala-εAhx-Lys; Phe-εAhx-εAhx-Lys, or p-aminophenylarsine oxidecoupled using EDC chemistry, to a ubiquitin recognition elementsselected from Arg-εAhx-Ala; Arg-β-Ala-εAhx-Ala; Arg-εAhx-εAhx-Ala;Phe-εAhx-Ala; Phe-β-Ala-εAhx-Ala; Phe-εAhx-εAhx-Ala. These compounds ofthe invention molecules are then added to the cells transfected with theECFP-Cys4 expression vector and subsequently treated with 100 ug/mlcyclohexamide to block further protein synthesis. The fluorescence ismeasured over time to determine the levels of the protein and theprotein bound to the compounds of the invention, using excitation at 440nm and emission at 480 nm to look at ECFP levels and 635 nm when the4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein based compounds are used.The stimulation of degradation seen with the p-aminophenylarsine oxidebased compounds was observed by drop in fluorescence of the ECFPrelative to control cells. In the case of the studies with the4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein based compounds aninitial rise of the FRET signal at 635 nm is seen followed by a drop inthe signal compared to controls where only4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein is used to treat thecells. In addition to these fluorescence studies, the levels of proteinusing western blot analysis is examined using an antibody to the ECFP,GFP (Clontech, Palo Alto, Calif.), which demonstrated that compounds ofthe invention lower the levels of the ECFP. Concentrations for thevarious compounds and other molecules used were from 0.1 uM to 100 uM.This study showed the ability to use the targeted ubiquitination toalter the levels and half-life of a protein in a living cell usingcompounds of the invention.

Control of Protein Levels in the Liver of a Transgenic Organism

[0217] An example of the above embodiment is the demonstration oftargeted ubiquitination to mediate quantitative and tissue specificcontrol of gene expression in transgenic mice. The expression vector wasconstructed using the luciferase gene and a liver specific promoterP_(LAP), the promoter of the liver-enriched activator protein drivingthe expression of the luciferase gene (Kistner A., 1996, Proc. Natl.Acad. Sci. 93, 10933-10938). The luciferase gene was engineered tocontain the AEAAAREACCRECCARA (SEQ ID NO 40), sequence at the C terminususing synthetic oligonucleotides and PCR based cloning. The finalexpression vector consisted of the P_(LAP), promoter driving theexpression of the luciferase gene containing the AEAAAREACCRECCARA (SEQID NO 49), sequence (the binding site for the compounds of theinvention). This expression vector was then used to generate transgenicmice. Transgenic mice lines were generated by pronuclear injection usingstandard techniques and analyzed by Southern blot using a BamHI-EcoRVfragment of the luciferase gene (Kistner A., 1996, Proc. Natl. Acad.Sci. 93, 10933-10938). The tissue specific expression of the modifiedluciferase gene was demonstrated using standard methods on liver,pancreas, kidney, stomach, muscle, thymus, heart, and tongue. In orderto modulate the levels of the luciferase gene the transgenic mice wereinjected with 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein coupledusing EDC chemistry, to a ubiquitin recognition elements selected fromArg-εAhx-Lys; Arg-β-Ala-εAhx-Lys; Arg-εAhx-εAhx-Lys; Phe-εAhx-Lys;Phe-β-Ala-εAhx-Lys; Phe-εAhx-εAhx-Lys, or p-aminophenylarsine oxidecoupled using EDC chemistry, to a ubiquitin recognition elementsselected from Arg-εAhx-Ala; Arg-β-Ala-εAhx-Ala; Arg-εAhx-εAhx-Ala;Phe-εAhx-Ala; Phe-βAla-εAhx-Ala; Phe-εAhx-εAhx-Ala, which formed a setof compounds of the invention. The serum concentrations achieved arefrom 1 micromolar to 1 millimolar. The levels of luciferase activity arelowered as the doses of the various compounds are increased. Thisresponse was also seen when the study was carried out using liver slicesinvitro using similar concentrations in the tissue culture medium usedfor the liver slice incubations.

[0218] Control of the Physiology a Transgenic Organism

[0219] An example of the above embodiment is the analysis of the effectof expressing CaMKII on specific forms of memory. CaMKII is aserine-threonine protein kinase expressed primarily in neurons of theforebrain. The ability of CaMKII to become persistently active inresponse to a transient Ca stimulus indicates its potential involvementin memory. Mutation of the Thr286 to Asp in CaMKII (CaMKII-Asp286)produces a calcium-independent form that mimics the auto-phosphorylatedform. The transgenic expression of CaMKII-Asp286 leads to a shift inresponse to stimulation as well as a severe defect in spatial memory. Toobtain tissue-specific and ubiquitin regulated degradation a line ofmice is generated expressing the CaMKII-Asp286 tagged with aAEAAAREACCRECCARA (SEQ ID NO 49), sequence (CaMKII-Asp286-tag) undercontrol of the native CaMKII promoter to ensure natural tissue specificexpression. In addition a line of mice was also constructed expressingbeta-galactosidase tagged with a AEAAAREACCRECCARA (SEQ ID NO 49),sequence (beta-gal-tag) under control of the native CaMKII promoter.These mice both demonstrated forebrain-specific expression. Severedefects in spatial memory were observed in response to CaMKII-Asp286-tagexpression using the Barnes circular maze. The treatment of these micebrains with the 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein,p-aminophenylarsine oxide coupled to a ubiquitin recognition elementsselected from Arg-εAhx-Cys; Arg-β-Ala-εAhx-Cys; Arg-εAhx-εAhx-Cys;Phe-εAhx-Cys; Phe-β-Ala-εAhx-Cys; Phe-εAhx-εAhx-Cys, demonstrated areversal of this profound memory impairment. In the mice with thebeta-gal-tag expression treatment of both the mice and tissues from thefore-brain with 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein,p-aminophenylarsine oxide coupled to a ubiquitin recognition elementsselected from Arg-εAhx-Cys; Arg-β-Ala-εAhx-Cys; Arg-εAhx-εAhx-Cys;Phe-εAhx-Cys; Phe-β-Ala-εAhx-Cys; Phe-εAhx-εAhx-Cys, demonstrateddramatic reductions in the levels of beta-galactosidase activity. Thissystem has the distinct advantage over the currently available systemswhich are based on multiple gene products, unnatural and modifiedpromoter elements, requiring multiple rounds of transfection and thescreening of multiple clonal cell lines to identify the desired cellline from each transfection; for example the Tet-Off™ and the Tet-On™gene expression system (U.S. Pat. No. 5,464,758) sold by Clontech (PaloAlto, Calif.; www.clontech.com).

[0220] Control of Gene Expression

[0221] In an extension of the above embodiments, relating to the controlof gene expression. The tag sequence or its equivalent can begenetically engineered into the coding sequence of various transcriptionand/or transactivating factors to render their protein levels within thecell sensitive to the presence of a small molecule activator of theubiquitination pathway. In this way any given transactivating factor (X)can be modified to contain tag sequence as above resulting in theexpression either in the native tissue or other-wise via themodification of said transactivating factors promoter and/or operatorand/or enhancer region, to allow the expression of X-tag. The levels ofthe X-tag protein can then be controlled via the use of a small moleculeactivator of the ubiquitination pathway in order to affect theexpression of any given gene dependent on said X for control and thusits protein product, in order to determine its role or to control someother aspect of the cell or organisms biochemistry, physiology or formthough the modification of gene expression. An example is atransactivating factor that controls multiple proteins expressionlevels. Control of this single transactivating factor results in theeffective control of multiple proteins via a small molecule activator ofthe ubiquitination pathway.

[0222] Control of Steroid Production in Genetically Engineered Animals

[0223] A ramification of the proceeding embodiment is the possibility ofgenerating modified cells and organisms which contain either a singleprotein or multiple proteins modified with a selective binding domainwhich allows the control of a specific gene with the cells or organismsto give rise to a desired biological effect. For example the reductionof boar taint in pigs can be achieved by the removal of the hormoneGnRH. This embodiment of the subject invention allows for themodification of the GnRH receptor to allow its targeted degradation inthe presence of a compound of the invention. In this way boar taint canbe controlled by feeding a compound of the invention, that downregulates the receptor resulting in the reduction of steroidbiosynthesis responsible for boar taint.

[0224] Control of Flower Color in Genetically Engineered Plants

[0225] In a further example, a gene for the biosynthesis of a flowercolor is modified allowing expression of a functional protein taggedwith the specific binding sequence. This expression of a modifiedprotein involved in the biosynthesis of flower color, such as the genesinvolved in the biosynthesis of flavonoids, carotenoids andanthocyanins; i.e. flavanone 3-hydroxylase, anthocyanin synthase,dihydroflavonol 4-reductase, flavonoid 3′,5′-hydroxylase, anthocyanin5-aromatic acyltransferase, UDP-glucose:flavonoid3-O-glucosyltransferase, anthocyanin rhamnosyltransferase, anthocyanin3′-methyltransferase, anthocyanin 3′5′-methyltransferase,leucoanthocyanidin dioxygenase, anthocyanidin synthase, anthocyaninacyltransferase, chalcone synthase, chalcone flavanone isomerase,glutathione S-transferase, allows for the modification of flower colorby addition of the compounds of the invention specific for the modifiedbiosynthetic protein. In addition to the proteins involved in thesynthesis of flower color, the gene products involved in the regulationof the expression of the synthases and other proteins involved in theproduction of flower color are also considered targets of the subjectinvention. Examples of the regulatory genes include the R and C1 genefamilies, an2 and jaf13, the delila gene. Quattrocchio F. 1998, Plant J.13(4) 475-488.

[0226] Resistance Control in Genetically Engineered Plants

[0227] In a still further example of the above embodiment of the subjectinvention relating to the selective control of protein levels to achievea desired biological response. The gene involved in the herbicideresistance to glyphosate (Roundup ®) in Roundup Ready ® soybeans theenolpyruvylshikimate-phosphate synthase from the bacteria agrobacteriumsp. Strain CP4 (CP4EPSPS), is engineered with a gene sequence encoding asmall molecule binding sequence i.e. tag as described above, whichallows the activation of the targeted degradation of the herbicideresistance marker using compounds of the invention. In this waytransgenic plants containing the engineered resistance gene CP4EPSPS canbe rendered sensitive to the herbicide glyphosate by contacting thetransgenic plants with compounds of the invention.

[0228] Gene Expression Control in Gene Therapy Vectors

[0229] In a further example of the selective control of protein levelsit is contemplated that the genes for pre-selected proteins areengineered to contain the coding sequence for a small molecules bindingsequence. Thus rendering the protein, expressed from the engineeredgenes of the pre-selected amino acid, targets for compounds of theinvention that allows these proteins activity and/or levels to becontrolled by the compounds of the invention. The engineered genes ofthe pre-selected amino acid are then cloned into vectors for genetransfer into a host.

[0230] In the case of human gene therapy vectors that are useful areviruses such as; adenovirus, retroviruses, herpes virus, vaccina virus.In the case of other organisms potential vectors for gene therapy areselected from the viruses which are known to infect these host or can bemodified to infect these hosts. In addition to these viral vectors whichoffer significant efficiencies, native DNA or RNA (not in the context ofa viral genome) are also useful for gene therapy. In the case of DNA thecloned gene for the pre-selected protein containing the smallmolecule-binding site is placed in the DNA sequence such that it isunder control of suitable transcription control elements. Thisengineered DNA is then administered to the organism in such a way thatDNA is taken up by cells efficiently resulting in the DNA being eithertranscribed and translated directly or integrated into the genomefollowed by transcription and translation. Typically DNA and RNA uptakeinto cells is poor and this is typically stimulated by the use ofvarious chemical and physical methods. Examples of chemical methods arethe use of liposomes, calcium phosphate, detergents, ion-exchangecompounds such as DEAE dextran, and also methods linked to specificreceptors such as the folate receptor via linkage to folate analogues.The physical methods that have proved valuable for getting DNA into acell are electroporation, heat, physical membrane perturbation such aspricking, and scrapping of cells.

[0231] Pharmaceutical Preparations of the Compounds of the Invention

[0232] The pharmacologically active compounds of the subject inventionsoptionally are combined with suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds. These are administered as tablets,dragees, capsules, and suppositories. The compositions are administered,for example, orally, rectally, vaginally, pulmonary or released throughthe buccal pouch of the mouth, and are optionally applied in solutionform by injection, orally or by topical administration such astransdermal patchs. The compositions may contain from about 0.1 to 99percent, preferably from about 50 to 90 percent, of the activecompound(s), together with the excipient(s).

[0233] For delivery of high molecular weight compounds and compoundswith poor bioavailability of the subject invention methods based onvarious known formulations and methods are contemplated; these includethe use of antibodies, pyridoxyl, insulin, transferrin, galactose,sialyl-LewisX, liposomes, asialolglycoprotein, folate, invasin,iontophoresis, galparan, transportan, homeobox peptides (such as thosebased on antennapedia residues 43-58), for intracellular delivery.

[0234] For parenteral administration by injection or intravenousinfusion, the active compounds are suspended or dissolved in aqueousmedium such as sterile water or saline solution. Injectable solutions orsuspensions optionally contain a surfactant agent such aspolyoxyethylenesorbitan esters, sorbitan esters, polyoxyethylene ethers,or solubilizing agents like propylene glycol or ethanol. The solutiontypically contains 0.01 to 5% of the active compounds. The activecompounds optionally are dissolved in pharmaceutical grade oils (ievegetable, synthetic) for intramuscular, sub-cutaneous or sub-dermalinjection. Such preparations contain about 1% to 50% of the activecompound(s) in oil. Also the active compounds optionally areincorporated into or onto particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, hydrogels, etc. orinto liposomes, niosomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, biodegradable injectable microcapsules ormicrospheres, or protein matrices, erythrocyte ghosts, spheroplasts,skin patches, or other known methods of releasing or packagingpharmaceuticals.

[0235] Suitable excipients include fillers such as sugars, for examplelactose, sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose and/or polyvinyl pyrrolidone.

[0236] Auxiliaries include flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate and/or polyethylene glycol. Dragee coresare provided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions are used,which optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsare optionally added to the tablets or dragee coatings, for example, foridentification or in order to characterize different compound doses.

[0237] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral useare obtained by combining the active compound(s) with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

[0238] Other pharmaceutical preparations which are useful for oraldelivery include push-fit capsules made of gelatin, as well assoft-sealed capsules made of gelatin and a plasticizer such as glycerolor sorbitol. The push-fit capsules contain the active compound(s) in theform of granules which optionally are mixed with fillers such aslactose, binders such as starches and/or lubricants such as talc ormagnesium stearate, and, optionally stabilizers. In soft capsules, theactive compounds are preferably dissolved or suspended in suitableliquids such as fatty oils, liquid paraffin, or polyethylene glycols. Inaddition, stabilizers optionally are added.

[0239] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water soluble form, forexample, water soluble salts. In addition, suspensions of the activecompounds as appropriate in oily injection suspensions are administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or tri-glycerides. Aqueous injection suspensions optionallyinclude substances which increase the viscosity of the suspension whichinclude, for example, sodium carboxymethylcellulose, sorbitol and/ordextran. The suspension optionally contains stabilizers.

[0240] In another embodiment, the active compounds are formulated aspart of a skin lotion for topical administration. Suitable lipophilicsolvents or vehicles include fatty oils, for example sesame oil orcoconut oil, or synthetic fatty acid esters, for example ethyl oleate ortriglycerides.

[0241] In another embodiment, the active compounds are formulated invehicles suitable for direct treatment of gastrointestinal mucosa.Examples include mouthwashes, liquids (solutions or suspensions) to beswallowed, or viscous fluids (e.g. solutions of methylcellulose,carboxymethylcellulose, xanthan gum, etc.) which are administered orallyor rectally.

[0242] Other pharmaceutical preparations which are used rectally,especially for treatment of the colon and rectum, include, for example,suppositories which consist of a combination of active compounds with asuppository base. Suitable suppository bases are, for example, naturalor synthetic triglycerides, paraffin hydrocarbons, polyethylene glycolsor higher alkanols. In addition, gelatin rectal capsules which consistof a combination of the active compounds with a base are useful. Basematerials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

[0243] Other pharmaceutical preparations which are used orally,especially for treatment of the lungs, trachea, sinus and oral cavity,include, for example, powders, foamates, nano-particles, liposomes,niosomes, microemulsions, micelles, unilamellar or multilamellarvesicles. These may optionally be administered as for example sprays andaerosols.

[0244] The following examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1

[0245] Targeted Degradation of HIV Integrase

[0246] Expression and Purification of His₆-HIV Integrase

[0247] Full length HIV-1 IN is expressed in E. coli and purified by theestablished protocols of Craigie, R., Hickman, A. B. and Engelman, A.(1995) in HIV Volume 2: A Practical Approach, pp53-71, J. Karn ed.,Oxford Univ. Press, New York. The pINSD.His plasmid, containing theHIV-1_(NL4-3) coding sequence inserted in the pET-15b His₆ expressionvector (Novagen), is available from the NIAID AIDS Research andReference Reagent Program as transformed HB101. pINSD.His is preparedusing a Qiagen plasmid purification kit, and transformed into BL21(DE3)by electroporation for expression in shaker flask cultures by Protocol2, from Craigie, R., Hickman, A. B. and Engelman, A. (1995) in HIVVolume 2: A Practical Approach, pp53-71, J. Karn ed., Oxford Univ.Press, New York. Following Protocol 4, as described in Craigie, R.,Hickman, A. B. and Engelman, A. (1995) in HIV Volume 2: A PracticalApproach, pp53-71, J. Karn ed., Oxford Univ. Press, New York, bacteriaare lysed and the His₆ HIV IN purified under native conditions. Theprotocol is basically a one step chelating column purification of a 2 MNaCl-soluble lysate fraction.

[0248] Synthesis of Bifunctional Trans-targeting Derivatives ofL-chicoric Acid

[0249] Compounds are designed based on bromoacetic acid derivatization,via ethylenediamine, of an L-chicoric acid carboxyl group for selectivereaction with thiols. Linkers are composed of aminocaproic acid andβ-alanine, variations of this are readily synthesized by solid phasemethods to include cysteine for conjugation to bromo acetylatedL-chicoric acid. Thiol addition to bromoacetic acid is selective,accomplished under mild reaction conditions, and yields are nearquantitative (Inman, J. K., Highet, P. F., Kolodny, N., and Robey, F. A.(1991) Bioconjugate Chem. 2, 458-463). A significant aspect of thisstrategy is that once L-chicoric acid has been successfullybromoacetylated and purified, any number of different trans-targetingcompounds are obtained easily and in high yield from recognition/linker“cassettes” generated readily from solid phase synthesis.

[0250] The synthesis of L-chicoric acid is accomplished followingliterature procedures (Panizzi, L., Scarpati, M. L. and Scarpati, R.(1954) Gazz. Chim. Ital. 84, 806-815, Scarpati, M. L. and Oriente, G.(1958) Tetrahedron 4, 43-48., FIG. 2). The bromoacetyl derivatizationstrategy is to generate bromoacetic acid anhydride for reaction withcommercially available Boc-blocked ethylenediamine (Aldrich) to giveN-bromoacetyl-ethylenediamine after TFA deprotection and crystallization(4, FIG. 3, Scheme 2). 4 is conjugated to L-chicoric acid activated withNHS at one of the symmetrically equivalent carboxylate groups. NHS esteractivation of acid groups for primary amine coupling is used routinelyin conjugation chemistry. Preparative RP HPLC is used for purificationof the desired monoester product from the reaction mixture. Theconjugation reaction to give the final bromoacetyl derivative ofL-chicoric acid, 6, is straightforward in terms of mixture complexityand product purification. NMR and mass spectral analysis monitor allsteps of the synthesis.

[0251] The E3α ubiquitination recognition elements are based on thestudies by Bachmair, A. and Varshavsky, A. (1989) Cell 56, 1019-1032.The aminocaproic acid (εAhx) and β-Ala will give the ubiquitinationrecognition elements considerably more degrees of freedom than a peptideand are not susceptible to proteinases. Combinations of aminocaproicacid and β-Ala are used to adjust hydrophobic character, flexibility andparticularly the length of the linkers. Changing between Type I (basic)and Type II (hydrophobic) recognition signals significantly affect thehydrophobic character of the trans-targeting compounds, but have aneffect on linker function since these sites are spatially distinct onE3α.

[0252] The first series of recognition/linkers include Arg (Type I) andPhe (Type II) recognition components and three different spacerelements; εAhx-Cys, β-Ala-εAhx-Cys, and εAhx-εAhx-Cys with molecularweights of approximately 315, 386 and 428, respectively.

[0253] Solid Phase Synthesis of E3α Recognition/Linker Components(Ubiquitination Recognition Elements)

[0254] Various ubiquitination recognition elements were synthesized bysolid phase peptide synthesis and characterized by C₁₈ reverse phaseHPLC and MALDI-TOF mass spectral analysis (American Peptide Company,Inc., Sunnyvale, Calif.). The linker elements include caproic acid(εAhx) and beta-alanine (β-Ala) for a high degree of freedom of motion,and a C-terminal Cys residue for specific thiol conjugation to targetingmolecule components. The compounds were synthesized to >90% purity in 10mg amounts. MW Arg-εAhx-Cys 390 Arg-β-Ala-εAhx-Cys 462 Arg-εAhx-εAhx-Cys521 Phe-εAhx-Cys 400 Phe-β-Ala-εAhx-Cys 452 Phe-εAbx-εAhx-Cys 531

[0255] Further ubiquitination recognition elements are synthesized asfollows using methods described above.

[0256] 1. Arg-Ala-εAhx-Cys

[0257] 2. Arg-Ala-β-Ala-εAhx-Cys

[0258] 3. Arg-Ala-εAhx-εAhx-Cys

[0259] 4. Phe-Ala-εAhx-Cys

[0260] 5. Phe-Ala-β-Ala-εAhx-Cys

[0261] 6. Phe-Ala-εAhx-εAhx-Cys

[0262] Synthesis of L-chicoric Acid

[0263]FIG. 2, Scheme 1.

[0264] To 0.36 g of caffeic acid (Aldrich) in 100 mL of H₂O is added 10g of sodium bicarbonate and the solution is cooled to 0° C. A 20%solution of COCl₂ in toluene (Fluka) is added slowly with stirring,followed by the slow addition of 20 mL of 6 M HCl The solid product isfiltered under vacuum, washed with H₂O and acetone, and recrystallizedfrom glacial acetic acid to give the blocked catechol of caffeic acid, 3(Panizzi, L., Scarpati, M. L. and Scarpati, R. (1954) Gazz. Chim. Ital.84, 806-815).

[0265] To 0.25 g of 3 in benzene is added 0.30 g of PCl₅ and thereaction mixture is refluxed until 20 min after complete solution, andthen allowed to stand for 1 hr. The solid product is rapidly filteredunder vacuum, washed with ether, and dried under vacuum to yield 4(Panizzi, L., Scarpati. M. L. and Scarpati, R. (1954) Gazz. Chim. Ital.84, 806-815).

[0266] A mixture of 0.23 g of 4 and 86 mg of L-tartaric acid (Aldrich)is heated on an oil bath under reduced pressure until fusion at 115° C.The reaction temperature is increased to 135° C. for 10 min, and thereaction is allowed to cool. The solid product is heated with 4.5 mL of80% acetic acid on a steam bath until dissolved, and then rotovapped.The residue is heated at 50° C. with 1.25 mL of H₂O, and the mixturefiltered to remove unreacted caffeic acid. The filtrate is extracted 2×with ether, and the ether layer is rotovapped. The residue is taken upinto H₂O with warming and adjusted to pH 6 with sodium bicarbonate.Caffeic acid is precipitated as a barium salt by the addition ofsaturated BaSO₄, collected and washed with 3% BaSO₄ bymicrocentrifugation, and then mixed with 0.75 mL 2 M HCl and 2 mL etheruntil in solution. The ether layer is removed and the aqueous phaseextracted 2× with ether. The combined ether extracts are dried overMgSO₄, rotovapped, and the product recrystallized from H₂O to yieldL-chicoric acid (Scarpatti and Oriente, 1958).

[0267] Symmetric Anhydride of Bromoacetic (Chloroacetic) Acid

[0268]FIG. 3, Scheme 2.

[0269] A pre-cooled 0.5 M solution of DCC in DCM (40 ml, 20 mmol) isadded to a stirred solution of bromoacetic acid (40 mmol) in DCM (20 ml)at 0° C. The reaction mixture is stirred for 30 min and filtered toremove the dicyclohexylurea that have formed, and the filtrate isevaporated on a rotary evaporator at 20° C. (Bioconjugate Chemistry1995, 6, 269).

[0270] N-bromoacetyl-N′-Boc-ethylenediamine (3)

[0271]FIG. 3, Scheme 2.

[0272] Freshly prepared bromoacetic anhydride (20 mmol) is dissolved in10 ml of acetonitrile, and the solution is added to a stirred solutionof N-Boc-ethylenediamine (18 mmol, Aldrich) and TEA (20 mmol) in THF (20ml) at 20° C. The progress of the reaction if followed by the ninhydrintest for free amines. When all Boc-ethylenediamine is consumed thereaction mixture is concentrated on a rotovap and dissolved in ethylacetate (150 ml). The solution is successively washed with 0.5 M sodiumbicarbonate (50 ml×2), 0.1 M sulfuric acid (50 ml×3), brine (50 ml×2),dried over sodium sulfate and concentrated providing the desiredN-bromoacetyl-N′-Boc-ethylenediamine (3).

[0273] N-bromoacetyl-ethylenediamine (4)

[0274]FIG. 3, Scheme 2.

[0275] N-bromoacetyl-N′-BOC-ethylenediamine (2 is dissolved in 50% TFAin dichloromethane (5 ml of the solution per mmol of 3) at 20° C. Thedeprotection is allowed to proceed for 30 min, then the reaction mixtureis concentrated on a rotary evaporator and solidifies upon addition ofdry ethyl ether. The solid material is filtered off, washed withether/petroleum ether on filter and dried. The desiredN-bromoacetyl-ethylenediamine is obtained in the form oftriflouroacetate salt.

[0276] Bromoacetylated Derivative of L-chicoric Acid (6)

[0277]FIG. 4, Scheme 3.

[0278] Mono NHS ester of L-chicoric acid (5, 0.1 mmol) is added to anexcess of the N-bromoacetyl-ethylenediamine (0.2 mmol) in a small volumeof THF in presence of DIEA (0.1 mmol). When all the activated ester isconsumed, the reaction mixture is diluted with ethyl acetate (150 ml),successively washed with 0.1 M sulfuric acid (100 ml×2) to remove theunreacted amine, brine (50 ml×2), dried over sodium sulfate andevaporated on a rotovap. 6 is further purified by crystallization fromappropriate solvents or by preparative RP HPLC.

[0279] Conjugation of 6 with the Recognition/Linkers;

[0280]FIG. 5, Scheme 4.

[0281] The recognition/linkers from solid phase synthesis (50 μmol) and6 (60 μmol) are dissolved in a small volume of 50 mM sodium acetatebuffer, pH 4.0 and purged with nitrogen. pH of the solution is raised upto 7-8 by addition of solid sodium bicarbonate. The reaction mixture isstirred at 20° C. until the Elman test shows absence of free thiols inthe mixture. The reaction mixture is diluted with 0.1% trifluoroaceticacid and the desired product is isolated by preparative RP HPLC (Ivanov,B., Grzesik, W., and Robey, F. A. (1995) Bioconjugate Chem. 6, 269-277).

[0282] In Vitro Reticulocyte Extract Assay for Targeted Degradation

[0283] Degradation is monitored using ¹²⁵I-labeled IN in a rabbitreticulocyte lysates by SDS-PAGE/auto radiography and by determinationof soluble ¹²⁵I after the precipitation of proteins with TCA. The use ofthis system to assess ubiquitin-dependent proteolysis is straightforwardand well established in the literature (Gonda, D. K., Bachmair, A.,Wunning, I., Tobias, J. W., Lane, W. S. and Varshavsky, A. (1989) J.Biol. Chem. 264, 16700-16712, Hershko, A., Ciechanover, A., Heller, H.,Haas, A. L., and Rose, I. A. (1980) Proc. Natl. Acad. Sci. USA 77,1783-1786).

[0284] The series of trans-targeting compounds are evaluated for theirability to initiate the degradation of [¹²⁵I]-IN in the reticulocytelysates. SDS-PAGE time course results show transitory multiubiquitinatedIN species, followed by loss of ¹²⁵I-labeled protein. The assay forTCA-soluble peptide product fragments is used to better quantitate ratesof degradation and effective concentrations.

[0285] Preparation of Rabbit Reticulocyte Lysates

[0286] Biocon, Inc. (Rockville, Md.) performed the induction andcollection of reticulocytes from NZW rabbits. A female NZW rabbitweighing, less than 2 kg was injected sub-cutaneous with 0.6 mL/kg of 20mg/mL phenylhydrazine on day 1,2,4 and 6. On day 8 the rabbit wasanesthetized with ketamine and bled out by heart bleed. The blood wascollected into heparinized tubes on ice, and washed 3 times with 5pellet volumes per wash of cold PBS. The reticulocyte lysates wereprepared by the addition of 1.5 volumes of cold H₂O 1 mM DTT per volumeof packed cells, followed by centrifugation for 2.5 hrs at 38,400× g.The supernatant was frozen in aliquots at −80 C. The reticulocyte lysatecan be used for 2 or 3 freeze/thaw cycles only.

[0287] TCA Precipitation Assay of [¹²⁵I]-protein Degradation inReticulocyte Lysates

[0288] Proteins were labeled by Lofstrand Laboratories Ltd.(Gaithersburg, Md.) Labeled to 0.12-0.50 μCi/μg by oxidation of Na¹²⁵Iusing an iodobead chloramine-T procedure.

[0289] The ubiquitin-dependent protein degradation assay was preformedby the addition of 70 μL of rabbit reticulocyte lysate and 5 μL of 0.06μCi/μL ¹²⁵I-labeled protein to 175 mL of reaction buffer containing 40mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP, 35 μg creatinephosphokinase (Sigma) and 10 mM phosphocreatine. The reaction were runat 37 C. in a heating block, and at time points 30 μL of the reactionwas transferred to 50 μL of cold 100 mg/mL BSA and protein wasprecipitated by the addition of 420 μL of 23% TCA followed by 15 min onice. The precipitated samples were microfuged for 2 min at 5000 rpm and300 μL of supernatant was then counted for TCA-soluble ¹²⁵I on a gammacounter (Hidex).

[0290] [¹²⁵I]-lysozyme(hen, Sigma), [¹²⁵I]-glutathione S-transferaseDegradation

[0291] The N-terminal sequences for lysozyme and GST samples submittedto Midwest Analytical were KVFGR and PPYTI, respectively. The only N-endrule stabilizing residues in mammalian cells are Gly, Val, Pro, and Met.Lysozyme is the usual positive control for the reticulocyte lysateassays; GST should be stable to N-end rule, ubiquitin-dependentproteolysis. Time points were taken every 30 min from 0 to 120 min. 25μL rxn samples counted directly in the gamma counter gave 55746 cpm forlysozyme and 45989 cpm for GST. Results demonstrated that the assay wasfunctional for the specific N-end rule degradation as described in theliterature. Time Lysozyme GST 0 1338 1320 30 7512 1474 60 11979 1723 9014976 1863 120 16337 2173

[0292] Table 1, Time Course of Lysozyme and Glutathione S-transferaseUbiquitin Mediated Degradation in the Reticulocyte Lysate

[0293] SDS-PAGE ¹²⁵I Protein Degradation Assay

[0294] The ubiquitin-dependent protein degradation assay was preformedby the addition of 70 μL of rabbit reticulocyte (or other cell) lysateand 5 μL of 0.06 μCi/μL ¹²⁵I-labeled protein to 175 mL of reactionbuffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP,35 μg creatine phosphokinase (Sigma) and 10 mM phosphocreatine. Thereaction was run at 37 C. in a heating block, and at time points 30 μLof the reaction are transferred to gel loading buffer. Samples are runon tricine 10-20% SDS-PAGE gels (Novex) for autoradiography on X-omatfilm (Kodak) to determine ¹²⁵I protein degradation.

Example 2

[0295] Selection, Discovery and/or Evaluation of UbiquitinationRecognition Elements

[0296] In order to determine if a given molecule or molecular element ispotentially valuable as a ubiquitination recognition element the assaydescribed above is run with [¹²⁵I]-lysozyme or other labeled proteinsubstrates in the presence of potential ubiquitination recognitionelements.

[0297] In the case of Arg-εAhx-Cys, Phe-εAhx-Cys these were run in thereticulocyte lysate using lysozyme and both demonstrated inhibition ofthe lysozyme degradation as expected for ubiquitination recognitionelements. The results at the 2 hour time point were 12,475 cpm for notreatment, 6,486 cpm for the 2mM Arg-εAhx-Cys treatment and 3,592 cpmfor the 5mM Phe-εAhx-Cys treatment. These results indicate that aubiquitination recognition element can be made from X-εAhx-linker whereX is an amino acid involved in the N-end recognition and the linker ischemistry which links this to a binding molecule for the target proteinof interest.

[0298] In an additional assay for ubiquitination recognition elementscompounds and peptides are added to HeLa or Jurkat cell extracts(Alkalay et al 1995, Proc. Natl. Acad. USA 92, 10599), containingradiolabeled IkappaB alpha or IkappaB beta, modulation of theubiquitination was monitored by gel electrophoresis of the labeledproteins. This allows the selection of ubiquitination recognitionelements specific for the ubiquitination pathway used for IkappaBdegradation (Yaron A, 1997, EMBO J. 16, 6486).

Example 3

[0299] Targeted Degradation of Glutathione S-transferase

[0300] Conjugation of Ubiquitination Recognition Elements to Glutathione(FIG. 6)

[0301] 4.18 mg (15.1 μmol) bismaleimidohexane (BMH, Pierce, Rockford,Ill.) in 200 μL of dimethylformamide was added slowly to 1.84 mg (6μmol) glutathione in 2 mL 20 mM potassium phosphate pH 7.0 The reactionwas followed by C₁₈ reverse phase HPLC. After 30 min at roomtemperature, the reaction mixture was centrifuged at 12,000 rpm for 2min to remove precipitate, and the sample was loaded onto a C₁₈ Sep-Pakcartridge pre-equilibrated with H₂O. The bound sample was washed with 2mL of 10% methanol/H₂O and eluted in 3 1mL fractions of 50% methanol.The second 1 mL product fraction was partially concentrated byevaporation of the methanol. This activated glutathione was then reactedwith the various ubiquitination recognition elements.

[0302] For example for Arg-εAhx-Cys the activated glutathione was addedto 50 μL of 20 mg/mL Arg-μAhx-Cys. The pH was adjusted to pH 6.5 by theaddition of 5 M sodium hydroxide and the reaction was followed by C₁₈reverse phase HPLC. This protocol repeated for the followingubiquitination recognition elements, Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys,Arg-εAhx-εAhx-Cys, Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys,KKERLLDDRHDSGLDSMKDEEC (SEQ ID NO 50) where the S in bold arephosphorylated, RAALAVLKSGNC (SEQ ID NO 51),HGFPPEVEEQDVGTLPISCAQESGMDRHC (SEQ ID NO 52). This generated a series ofcompounds for testing in the rabbit reticulocyte, HeLa cell and Jurkatcell lysates.

[0303] Glutathione S-transferase (Sigma, St. Louis, Mo.) was labeled byLofstrand Laboratories Ltd. (Gaithersburg, Md.) Labeled to 0.12-0.50μCi/μg by oxidation of Na¹²⁵I using an iodobead chloramine-T procedure.

[0304] The ubiquitin-dependent protein degradation assay was preformedby the addition of 70 μL of rabbit reticulocyte (or other cell) lysateand 5 μL of 0.06 μCi/μL ¹²⁵I-labeled Glutathione S-transferase to 175 mLof reaction buffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂,0.5 mM ATP, 35 μg creatine phosphokinase (Sigma) and 10 mMphosphocreatine. To demonstrate the targeted degradation of the GST,various concentrations of the compounds from the above synthesis wereadded to the lysate (10 to 0.001 mM). The reaction were run at 37 C. ina heating block, and at time points 30 μL of the reaction wastransferred to 50 μL of cold 100 mg/mL BSA and protein was precipitatedby the addition of 420 μL of 23% TCA followed by 15 min on ice. Theprecipitated samples were microfuged for 2 min at 5000 rpm and 300 μL ofsupernatant was then counted for TCA-soluble ¹²⁵I on a gamma counter(Hidex).

[0305] Time points are taken every 30 min from 0 to 120 min. Resultsdemonstrate targeted degradation.

Example 4

[0306] Targeted Degradation of Anti Fluorescein Antibody

[0307] Conjugation of Ubiquitination Recognition Elements toFluorescein-5-maleimide (FIG. 7)

[0308] Arg-εAhx-Cys. To 400 μL of 5 mg/mL Arg-εAhx-Cys (2.00 mg, 5.13μmol) was added 219 μL of 50 mg/mL fluorescein-5-maleimide indimethylformamide (Pierce, 10.95 mg, 25.65 μmol, 5-fold molar excess)and the pH was adjusted to pH 6.5 with 5 M sodium hydroxide. Thereaction was followed by C₁₈ reverse phase HPLC. After 60 min at roomtemperature, the sample was loaded onto a C₁₈ Sep-Pak cartridgepre-equilibrated with H₂O. The bound sample was washed with 2 mL of 10%methanol/H₂O and the product eluted in 3 1 mL fractions of 60% methanol.This protocol is repeated for the following ubiquitination recognitionelements, Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys, Arg-εAhx-εAhx-Cys,Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-Ahx-εAhx-Cys.KKERLLDDRHDSGLDSMKDEEC (SEQ ID NO 50) where the S in bold arephosphorylated, RAALAVLKSGNC (SEQ ID NO 51),HGFPPEVEEQDVGTLPISCAQESGMDRHC (SEQ ID NO 52). This generated a series ofcompounds for testing in the rabbit reticulocyte lysate.

[0309] Anti fluorescein antibodies (Fitzgerald and Molecular Probes, OR)was labeled by Lofstrand Laboratories Ltd. (Gaithersburg, Md.) Labeledto 0.12-0.50 μCi/μg by oxidation of Na¹²⁵I using an iodobeadchloramine-T procedure.

[0310] The ubiquitin-dependent protein degradation assay was preformedby the addition of 70 μL of rabbit reticulocyte (or other cell) lysateand 5 μL of 0.06 μCi/μL ¹²⁵I-labeled anti fluorescein antibody to 175 mLof reaction buffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂,0.5 mM ATP, 35 μg creatine phosphokinase (Sigma) and 10 mMphosphocreatine. To demonstrate the targeted degradation of the antifluorescein antibodies, various concentrations of the compounds from theabove synthesis were added to the lysate (10 to 0.001 mM). The reactionwere run at 37 C. in a heating block, and at time points 30 μL of thereaction was transferred to 50 μL of cold 100 mg/mL BSA and protein wasprecipitated by the addition of 420 μL of 23% TCA followed by 15 min onice. The precipitated samples were microfuged for 2 min at 5000 rpm and300 μL of supernatant was then counted for TCA-soluble ¹²⁵I on a gammacounter (Hidex). Time points are taken every 30 min from 0 to 120 min.Results demonstrate the targeted degradation.

Example 5

[0311] Targeted Degradation of Thioredoxin

[0312] Conjugation of Ubiquitination Recognition Elements to4-aminophenyl Arsenoxide.

[0313] Arg-ε-Ahx-Cys. To 1.83 mg (10 μmol) 4-aminophenyl arsenoxide in100 μL dimethylformamide was added 5.3 μL of 50 mg/mL ethyleneglycobis(sulfo-succinimidylsuccinate) (Pierce, 15 μmol, 1.5 equivalents)in dimethyformarnide. After 30 min reaction time at room temperature,2.0 mg (5.13 μmol) Arg-εAhx-Lys in 400 μL 20 mM potassium phosphate pH6.5. The reaction was followed by C₁₈ reverse phase HPLC. After 30 min,the derivatized peptide product was separated from the reaction mixtureby C₁₈ Sep-Pak solid phase extraction. The bound sample was washed with2 mL of 10% methaol/H₂O and eluted in 3 1 mL fractions of 60% methanol.This protocol is repeated for the following ubiquitination recognitionelements, Arg-εAhx-Lys, Arg-β-Ala-εAhx-Lys, Arg-εAhx-εAhx-Lys,Phe-εAhx-Lys, Phe-β-Ala-εAhx-Lys, Phe-εAhx-εAhx-Lys,KAADADEWCDSGLGSLGPDA (SEQ ID NO 42) where the S in bold arephosphorylated, RHALDDVSNK (SEQ ID NO 54), HGFPPEVEEQDVGTLPISCAQESGMDRHK(SEQ ID NO 55). This generated a series of compounds for testing in therabbit reticulocyte lysate.

[0314] Thioredoxin was prepared following standard method from theplasmid vector pBAD/Thio, (Invitrogen, Carlsbad, Calif.). The plasmidvector was transformed into TOP10 cells and colonies grown up in LB with50 micrograms/ml ampicillin overnight at 37 C. This overnight culturewas then used to inoculate a large culture of LB with 50 micrograms/mlampicillin and supplemented with arabinose to induce expression. Theculture was then harvested and lysed by sonication and run on to aProBond™ column (Invitrogen), following the manufactures protocol toyield purified thioredoxin.

[0315] Thioredoxin was labeled by Lofstrand Laboratories Ltd.(Gaithersburg, Md.) Labeled to 0.12-0.50 μCi/μg by oxidation of Na¹²⁵Iusing an iodobead chloramine-T procedure.

[0316] The ubiquitin-dependent protein degradation assay was preformedby the addition of 70 μL of rabbit reticulocyte lysate and 5 μL of 0.06μCi/μL ¹²⁵I-labeled thioredoxin to 175 mL of reaction buffer containing40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP, 35 μg creatinephosphokinase (Sigma) and 10 mM phosphocreatine. To demonstrate thetargeted degradation of the thioredoxin, various concentrations of thecompounds from the above synthesis were added to the lysate (10 to 0.001mM). The reaction were run at 37 C. in a heating block, and at timepoints 30 μL of the reaction was transferred to 50 μL of cold 100 mg/mLBSA and protein was precipitated by the addition of 420 μL of 23% TCAfollowed by 15 min on ice. The precipitated samples were microfuged for2 min at 5000 rpm and 300 μL of supernatant was then counted forTCA-soluble ¹²⁵I on a gamma counter (Hidex).

[0317] Time points are taken every 30 min from 0 to 120 mm. Resultsdemonstrate the targeted degradation.

[0318] It will be readily apparent to those skilled in the art thatnumerous modifications and additions may be made to both the presentinvention without departing from the invention disclosed.

1 67 1 20 PRT Unknown Organism Description of Unknown Organism PESTexample sequence 1 Met Glu Phe Met His Ile Ser Pro Pro Glu Pro Glu SerGlu Glu Glu 1 5 10 15 Glu Glu His Ser 20 2 10 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 2 Met Glu Phe MetHis Glu Ser His Ser Ser 1 5 10 3 16 PRT Unknown Organism Description ofUnknown Organism PEST example sequence 3 Met Glu Phe Met His Ile Ser ProPro Glu Pro Glu Ser His Ser Ser 1 5 10 15 4 15 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 4 Met Glu Phe MetHis Glu Ser Glu Glu Glu Glu Glu His Ser Ser 1 5 10 15 5 10 PRT UnknownOrganism Description of Unknown Organism PEST example sequence 5 Met GluAla Ser Glu Glu Glu Glu Glu Phe 1 5 10 6 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 6 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro 1 5 10 15 Met Ser CysAla Gln Glu Ser Gly Met Asp Arg His 20 25 7 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 7 His Gly Phe ProPro Ala Val Ala Ala Gln Asp Asp Gly Thr Leu Pro 1 5 10 15 Met Ser CysAla Gln Glu Ser Gly Met Asp Arg His 20 25 8 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 8 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Asp Gly Ala Leu Pro 1 5 10 15 Met Ser CysAla Gln Glu Ser Gly Met Asp Arg His 20 25 9 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 9 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu Pro 1 5 10 15 Met Ser CysAla Gln Glu Ser Gly Met Asp His His 20 25 10 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 10 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Val Gly Thr Leu Pro 1 5 10 15 Met Ser CysAla Gln Glu Ser Gly Met Asp Arg His 20 25 11 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 11 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Val Gly Thr Leu Pro 1 5 10 15 Ile Ser CysAla Gln Glu Ser Gly Met Asp Arg His 20 25 12 28 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 12 His Gly Phe ProPro Glu Val Glu Glu Gln Asp Ala Ser Thr Leu Pro 1 5 10 15 Val Ser CysAla Trp Glu Ser Gly Met Lys Arg His 20 25 13 26 PRT Unknown OrganismDescription of Unknown Organism PEST example sequence 13 Phe Pro Pro GlyVal Glu Glu Pro Asp Val Gly Pro Leu Pro Val Ser 1 5 10 15 Cys Ala TrpGlu Ser Gly Met Lys Arg His 20 25 14 27 PRT Unknown Organism Descriptionof Unknown Organism PEST example sequence 14 Phe Leu Ala Glu Val Glu GluGln Asp Val Ala Ser Leu Pro Leu Ser 1 5 10 15 Cys Ala Cys Glu Ser GlyIle Glu Tyr Pro Ala 20 25 15 25 PRT Artificial Sequence Description ofArtificial Sequence consensus sequence 15 Phe Xaa Xaa Glu Val Glu GluGln Asp Xaa Xaa Xaa Leu Pro Xaa Ser 1 5 10 15 Cys Ala Xaa Glu Ser GlyXaa Xaa Xaa 20 25 16 26 PRT Artificial Sequence Description ofArtificial Sequence consensus sequence 16 Phe Xaa Xaa Ala Val Ala AlaGln Asp Xaa Xaa Xaa Leu Pro Xaa Ser 1 5 10 15 Cys Ala Xaa Glu Ser GlyXaa Xaa Xaa Xaa 20 25 17 28 PRT Artificial Sequence Description ofArtificial Sequence consensus sequence 17 His Gly Xaa Xaa Pro Glu ValXaa Xaa Xaa Asp Xaa Xaa Xaa Leu Xaa 1 5 10 15 Xaa Ser Cys Ala Gln GluSer Gly Met Xaa Xaa Xaa 20 25 18 9 PRT Unknown Organism Description ofUnknown Organism D box example sequence 18 Arg His Ala Leu Asp Asp ValSer Asn 1 5 19 9 PRT Unknown Organism Description of Unknown Organism Dbox example sequence 19 Arg Leu Ala Leu Asn Asn Val Thr Asn 1 5 20 9 PRTUnknown Organism Description of Unknown Organism D box example sequence20 Arg Ala Ala Leu Gly Asp Val Ser Asn 1 5 21 9 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 21 Arg Gln ValLeu Gly Asp Ile Gly Asn 1 5 22 9 PRT Unknown Organism Description ofUnknown Organism D box example sequence 22 Arg Ala Ala Leu Gly Asp LeuGln Asn 1 5 23 9 PRT Unknown Organism Description of Unknown Organism Dbox example sequence 23 Arg Ala Ala Leu Gly Asn Ile Ser Asn 1 5 24 9 PRTUnknown Organism Description of Unknown Organism D box example sequence24 Arg Asn Thr Leu Gly Asp Ile Gly Asn 1 5 25 9 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 25 Arg Thr AlaLeu Gly Asp Ile Gly Asn 1 5 26 9 PRT Unknown Organism Description ofUnknown Organism D box example sequence 26 Arg Ala Ala Leu Gly Glu IleGly Asn 1 5 27 9 PRT Unknown Organism Description of Unknown Organism Dbox example sequence 27 Arg Ala Val Leu Glu Glu Ile Gly Asn 1 5 28 9 PRTUnknown Organism Description of Unknown Organism D box example sequence28 Arg Ser Ala Phe Gly Asp Ile Thr Asn 1 5 29 9 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 29 Arg Ser IleLeu Gly Val Ile Gln Ser 1 5 30 9 PRT Unknown Organism Description ofUnknown Organism D box example sequence 30 Arg Ala Ala Leu Gly Val IleThr Asn 1 5 31 10 PRT Unknown Organism Description of Unknown Organism Dbox example sequence 31 Arg Thr Val Leu Gly Val Ile Gly Asp Asn 1 5 1032 9 PRT Unknown Organism Description of Unknown Organism D box examplesequence 32 Arg Thr Val Gly Val Leu Gln Glu Asn 1 5 33 9 PRT UnknownOrganism Description of Unknown Organism D box example sequence 33 ArgAla Ala Leu Gly Thr Val Gly Glu 1 5 34 10 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 34 Arg Thr ValLeu Gly Val Leu Thr Glu Asn 1 5 10 35 11 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 35 Arg Ala AlaLeu Ala Val Leu Lys Ser Gly Asn 1 5 10 36 9 PRT Unknown OrganismDescription of Unknown Organism D box example sequence 36 Arg Leu ProLeu Ala Ala Lys Asp Asn 1 5 37 9 PRT Unknown Organism Description ofUnknown Organism D box example sequence 37 Arg Gln Leu Phe Pro Ile ProLeu Asn 1 5 38 9 PRT Unknown Organism Description of Unknown Organism Dbox example sequence 38 Arg Arg Thr Leu Lys Val Ile Gln Pro 1 5 39 9 PRTUnknown Organism Description of Unknown Organism D box general structure39 Arg Xaa Ala Leu Gly Xaa Xaa Xaa Asn 1 5 40 21 PRT Unknown OrganismDescription of Unknown Organism ubiquitination recognition element 40Lys Glu Phe Ala Val Pro Asn Glu Thr Ser Asp Ser Gly Phe Ile Ser 1 5 1015 Gly Pro Gln Ser Ser 20 41 22 PRT Unknown Organism Description ofUnknown Organism ubiquitination recognition element 41 Lys Gly Pro AspGlu Ala Glu Glu Ser Gln Tyr Asp Ser Gly Leu Glu 1 5 10 15 Ser Leu ArgSer Leu Arg 20 42 20 PRT Unknown Organism Description of UnknownOrganism ubiquitination recognition element 42 Lys Ala Ala Asp Ala AspGlu Trp Cys Asp Ser Gly Leu Gly Ser Leu 1 5 10 15 Gly Pro Asp Ala 20 4321 PRT Unknown Organism Description of Unknown Organism ubiquitinationrecognition element 43 Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp SerGly Leu Asp Ser 1 5 10 15 Met Lys Asp Glu Glu 20 44 14 PRT ArtificialSequence Description of Artificial Sequence consensus sequence 44 LysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Ser Gly 1 5 10 45 12 PRTUnknown Organism Description of Unknown Organism ubiqitinationrecognition element 45 Ser Tyr Leu Asp Ser Gly Ile His Ser Gly Ala Thr 15 10 46 12 PRT Unknown Organism Description of Unknown Organismubiqitination recognition element 46 Arg Ala Glu Asp Ser Gly Asn Glu SerGlu Gly Glu 1 5 10 47 6 PRT Unknown Organism Description of UnknownOrganism example eptide 47 Cys Cys Xaa Xaa Cys Cys 1 5 48 17 PRT UnknownOrganism Description of Unknown Organism example peptide 48 Trp Glu AlaAla Ala Arg Glu Ala Cys Cys Arg Glu Cys Cys Ala Arg 1 5 10 15 Ala 49 17PRT Unknown Organism Description of Unknown Organism example peptide 49Ala Glu Ala Ala Ala Arg Glu Ala Cys Cys Arg Glu Cys Cys Ala Arg 1 5 1015 Ala 50 22 PRT Unknown Organism Description of Unknown Organismubiquitination recognition element 50 Lys Lys Glu Arg Leu Leu Asp AspArg His Asp Ser Gly Leu Asp Ser 1 5 10 15 Met Lys Asp Glu Glu Cys 20 5112 PRT Unknown Organism Description of Unknown Organism ubiquitinationrecognition element 51 Arg Ala Ala Leu Ala Val Leu Lys Ser Gly Asn Cys 15 10 52 29 PRT Unknown Organism Description of Unknown Organismubiquitination recognition element 52 His Gly Phe Pro Pro Glu Val GluGlu Gln Asp Val Gly Thr Leu Pro 1 5 10 15 Ile Ser Cys Ala Gln Glu SerGly Met Asp Arg His Cys 20 25 53 9 PRT Artificial Sequence Descriptionof Artificial Sequence consensus sequence 53 Arg Xaa Xaa Leu Gly Xaa IleXaa Asn 1 5 54 10 PRT Unknown Organism Description of Unknown Organismubiquitination recognition element 54 Arg His Ala Leu Asp Asp Val SerAsn Lys 1 5 10 55 29 PRT Unknown Organism Description of UnknownOrganism ubiquitination recognition element 55 His Gly Phe Pro Pro GluVal Glu Glu Gln Asp Val Gly Thr Leu Pro 1 5 10 15 Ile Ser Cys Ala GlnGlu Ser Gly Met Asp Arg His Lys 20 25 56 4 PRT Unknown OrganismDescription of Unknown Organism binding peptide 56 Tyr Glu Glu Ile 1 5718 PRT Unknown Organism Description of Unknown Organism binding peptide57 Asp Arg Glu Gly Cys Arg Arg Gly Trp Val Gly Gln Cys Lys Ala Trp 1 510 15 Phe Asn 58 22 PRT Unknown Organism Description of Unknown Organismbinding peptide 58 Glu Thr Pro Thr Phe Thr Trp Glu Glu Ser Asn Ala TyrTyr Trp Gln 1 5 10 15 Pro Tyr Ala Leu Pro Leu 20 59 12 PRT UnknownOrganism Description of Unknown Organism binding peptide 59 Thr Phe ValTyr Trp Gln Pro Tyr Ala Leu Pro Leu 1 5 10 60 15 PRT Unknown OrganismDescription of Unknown Organism binding peptide 60 Val Ser Leu Ala ArgArg Pro Leu Pro Pro Leu Pro Gly Gly Lys 1 5 10 15 61 17 PRT UnknownOrganism Description of Unknown Organism binding peptide 61 Lys Gly GlyGly Ala Ala Pro Pro Leu Pro Pro Arg Asn Arg Pro Arg 1 5 10 15 Leu 62 15PRT Unknown Organism Description of Unknown Organism binding peptide 62Ala Glu Cys His Pro Gln Gly Pro Pro Cys Ile Glu Gly Arg Lys 1 5 10 15 6313 PRT Unknown Organism Description of Unknown Organism binding peptide63 Gly Ala Cys Arg Arg Glu Thr Ala Trp Ala Cys Gly Ala 1 5 10 64 12 PRTUnknown Organism Description of Unknown Organism binding peptide 64 AspIle Thr Trp Asp Gln Leu Trp Asp Leu Met Lys 1 5 10 65 13 PRT UnknownOrganism Description of Unknown Organism binding peptide 65 Arg Asn MetSer Trp Leu Glu Leu Trp Glu His Met Lys 1 5 10 66 4 PRT Unknown OrganismDescription of Unknown Organism ubiquitination recognition element 66Arg Ala Ala Cys 1 67 4 PRT Unknown Organism Description of UnknownOrganism ubiquitination recognition element 67 Pro Ala Ala Cys 1

What is claimed is:
 1. A compound for activating the ubiquitination of atarget protein comprising; a) a ubiquitination recognition element whichis able to bind to either the E3 or E2 functional elements of theubiquitination system, wherein said ubiquitination recognition elementhas a molecular weight less than 30,000 and has a binding affinity forsaid E3 and/or E2 elements of the ubiquitination system of at least 10²M⁻¹ and; a target protein binding element that is able to bindspecifically to a target protein wherein said target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for said target protein greater than 10⁵ M⁻¹, wherein saidubiquitination recognition element is covalently linked to said targetprotein binding element.
 2. A compound for activating the ubiquitinationof a target protein comprising; a) a ubiquitination recognition peptideelement which is able to bind to either the E3 or E2 functional elementsof the ubiquitination system, wherein said ubiquitination recognitionpeptide element has a molecular weight less than 30,000 and has abinding affinity for said E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹ and; b) a target protein binding element thatis able to bind specifically to a target protein wherein said targetprotein binding element has a molecular weight of less than 30,000 andhas a binding affinity for said target protein greater than 10⁵M⁻¹,wherein said ubiquitination recognition peptide element is covalentlylinked to said target protein binding element.
 3. A compound foractivating the ubiquitination of a target protein comprising; a) aubiquitination recognition element which is able to bind to either theE3 or E2 functional elements of the ubiquitination system, wherein saidubiquitination recognition element has a molecular weight less than30,000 and has a binding affinity for said E3 and/or E2 elements of theubiquitination system of at least 10² M⁻¹ and; b) a target proteinbinding peptide element that is able to bind specifically to a targetprotein wherein said target protein binding peptide element has amolecular weight of less than 30,000 and has a binding affinity for saidtarget protein greater than 10⁵ M⁻¹, wherein said ubiquitinationrecognition element is covalently linked to said target protein bindingpeptide element.
 4. A compound for activating the ubiquitination of atarget protein comprising; a) a ubiquitination recognition peptideelement which is able to bind to either the E3 or E2 elements of theubiquitination system, wherein said ubiquitination recognition peptideelement has a molecular weight less than 30,000 and has a bindingaffinity for said E3 and/or E2 elements of the ubiquitination system ofat least 10² M⁻¹ and; b) a target protein binding peptide element thatis able to bind specifically to a target protein wherein said targetprotein binding peptide element has a molecular weight of less than30,000 and has a binding affinity for said target protein greater than10⁵ M⁻¹, wherein said ubiquitination recognition peptide element iscovalently linked to said target protein binding peptide element.
 5. Acompound as in claim 1 wherein said ubiquitination recognition elementhas an affinity of at least 10³ M⁻¹ and a molecular weight between 50and 10,000.
 6. A compound as in claim 5 wherein said target proteinbinding element has a molecular weight from 50 to 10,000 and a bindingaffinity of greater than 10⁶ M⁻¹.
 7. A compound as in claim 1 whereinsaid ubiquitination recognition element has an affinity of at least 10⁴M⁻¹ and a molecular weight between 50 and 3,000.
 8. A compound as inclaim 1 wherein said target protein binding element has a molecularweight from 50 to 3,000 and a binding affinity of greater than 10⁷ M⁻¹.9. A compound as in claim 5 wherein said target protein binding elementhas a molecular weight from 50 to 3,000 and a binding affinity ofgreater than 10⁸ M⁻¹.
 10. A compound as in claim 1 wherein saidubiquitination recognition element contains an amino acid with a freeamino terminal selected from the group consisting of Phe, Arg, Lys, Trp,Leu, Asn, Asp, Gln, Tyr, His, Glu, Cys, Thr, Ser and Ala and oxidizedderivatives thereof.
 11. A compound as in claim 1 wherein saidubiquitination recognition element contains an amino acid selected fromthe group consisting of Phe, Arg, Lys, Asn, Asp, Gln, Glu and Cys.
 12. Acompound as in claim 1 wherein said ubiquitination recognition elementcontains an amino acid selected from the group consisting of Arg, Phe,Asp, Gin and Glu.
 13. A compound as in claim 1 wherein saidubiquitination recognition element contains a moiety selected from thegroup consisting of Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys, Arg-εAhx-εAhx-Cys,Phe-ε-Ahx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys, Arg-Ala-εAhx-Cys,Arg-Ala-β-Ala-εAhx-Cys, Arg-Ala-εAhx-εAhx-Cys, Phe-Ala-εAhx-Cys,Phe-Ala-β-Ala-εAhx-Cys and Phe-Ala-εAhx-εAhx-Cys.
 14. A compound as inclaim 1 wherein said ubiquitination recognition element contains amoiety selected from the group consisting of; Arg-εAhx-Cys,Arg-β-Ala-εAhx-Cys, Arg-εAhx-εAhx-Cys, Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys,Phe-εAhx-εAhx-Cys.
 15. A compound as in claim 1 wherein said recognitionelement contains a moiety selected from the group consisting ofPhe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys.
 16. A compound asin claim 1 wherein said ubiquitination recognition element is a compoundable to inhibit a ubiquitination reaction by binding to a recognitionsite of a ubiquitination system.
 17. A compound as in claim 1 whereinsaid ubiquitination recognition element is a compound able to interactwith the recognition site of the ubiquitination system, said recognitionsites selected from the recognition sites for a ubiquitinationrecognition signal selected from the group consisting of N-endN-recognin, ‘destruction box’ or D box, PEST motifs, Deg1, Deg 2, delta(δ) domains, WW domain binding peptides and phosphorylated sequences.18. A compound as in claim 17 wherein said ubiquitination recognitionelement is a compound able to inhibit a ubiquitination reaction bybinding to the recognition site of the ubiquitination system, saidrecognition sites selected from the recognition sites for aubiquitination recognition signal selected from the group consisting ofN-end N-recognin, ‘destruction box’ or D box, phosphorylated sequences.19. A compound as in claim 1 wherein said ubiquitination recognitionelement is a compound able to inhibit a ubiquitination reaction bybinding to the recognition site of the ubiquitination system, whereinsaid ubiquitination system is the N-end rule ubiquitination system. 20.A compound as in claim 1 wherein said degradation results in alteredpresentation of degradation products on MHC proteins.
 21. A compound asin claim 20 wherein said MHC proteins are selected from MHC class I andMHC class II.
 22. A compound as in claim 1 wherein said compound has amolecular weight of less than 3,000.
 23. A compound as in claim 1wherein said ubiquitination recognition element binds the sameubiquitination recognition site as an N-recognin or its equivalent. 24.A method of modulating the level and/or activity of at least one targetprotein in an eukaryotic cell via the modulation of ubiquitination ofsaid at least one target protein comprising contacting said cell with acompound comprising; a) a ubiquitination recognition element which isable to bind to either the E3 or E2 elements of the ubiquitinationsystem, wherein said ubiquitination recognition element has a molecularweight less than 30,000 and has a binding affinity for said E3 and/or E2elements of the ubiquitination system of at least 10² M⁻¹ and; b) atarget protein binding element that is able to bind specifically to atarget protein wherein said target protein binding element has amolecular weight of less than 30,000 and has a binding affinity for saidtarget protein greater than 10⁵ M⁻¹, wherein said ubiquitinationrecognition element is covalently linked to said target protein bindingelement.
 25. The method of claim 24 where said at least one targetprotein is modulated to cause a physiological or metabolic change. 26.The method of claim 24 where said at least one target protein ismodulated to cause a pharmacological change.
 27. The method of claim 24where said at least one target protein is modulated to treat a disease.28. The method of claim 24 where said contacting said cell is achievedby administering said compound to a mammal.
 29. The method of claim 28where said at least one target protein is an antigen.
 30. A method as inclaim 29 wherein said mammal is a human.
 31. A method of treating aninfection in a mammal comprising administering to said mammal an amountof a compound sufficient to eliminate and/or reduce said infection saidcompound comprising; a) a ubiquitination recognition element which isable to bind to either the E3 or E2 elements of the ubiquitinationsystem, wherein said ubiquitination recognition element has a molecularweight less than 30,000 and has a binding affinity for said E3 and/or E2elements of the ubiquitination system of at least 10² M⁻¹ and; b) atarget protein binding element that is able to bind specifically to atarget protein wherein said target protein binding, element has amolecular weight of less than 30,000 and has a binding affinity for saidtarget protein greater than 10⁵ M⁻¹, wherein said ubiquitinationrecognition element is covalently linked to said target protein bindingelement.
 32. The method of claim 31 wherein said infection is a viralinfection.
 33. The method of claim 31 wherein said infection is causedby a virus selected from the group consisting of hepatitis A, hepatitisB, hepatitis C, hepatitis G, HIV1, HIV2, Herpes, CMV, rabies, RSV. 34.The method of claim 31 wherein said infection is caused by a parasiticinfection.
 35. The method of claim 31 wherein said infection is causedby an eukaryotic organism.
 36. A method of selectively targetingubiquitination in a cell comprising contacting said cell with a compoundas in claim
 1. 37. The method of claim 36 where said ubiquitinationrecognition element is recognized by an E3 for the N-end rule.
 38. Amethod of treating a tumor in a mammal comprising administering to saidmammal an amount of a compound sufficient to reduce the size of saidtumor, said compound comprising; a) a ubiquitination recognition elementwhich is able to bind to either the E3 or E2 elements of theubiquitination system, wherein said ubiquitination recognition elementhas a molecular weight less than 30,000 and has a binding affinity forsaid E3 and/or E2 elements of the ubiquitination system of at least 10²M⁻¹ and; b) a target protein binding element that is able to bindspecifically to a target protein wherein said target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for said target protein greater than 10⁵ M⁻¹, wherein saidubiquitination recognition element is covalently linked to said targetprotein binding element.
 39. A method of generating a compound foractivating ubiquitination of a target protein which comprises covalentlylinking a target protein binding element to a ubiquitination recognitionelement.
 40. A method as in claim 24 wherein said compound activates theubiquitination of a protein bound to said target protein.
 41. A methodfor controlling pests, comprising administering to said pests aneffective dose of the compound of claim
 1. 42. A ubiquitinationrecognition element comprising at least one structural element selectedfrom the group consisting of compound Z, Arg-εAhx-linker,Arg-β-Ala-εAhx-linker, Arg-εAhx-εAhx-linker, Phe-εAhx-linker,Phe-β-Ala-εAhx-linker, Phe-εAhx-εAhx-linker, Arg-Ala-εAhx-linker,Arg-Ala-β-Ala-εAhx-linker, Arg-Ala-ΕAhx-εAhx-linker,Phe-Ala-εAhx-linker, Phe-Ala-β-Ala-εAhx-linker,Phe-Ala-εAhx-εAhx-linker.